4′-C-ethynyl pyrimidine nucleoside compounds and pharmaceutical compositions

ABSTRACT

The invention provides 4′-C-ethynyl pyrimidine nucleosides (other than 4′-C-ethynylthymidine) represented by formula [I]:                    
     wherein B represents a base selected from the group consisting of pyrimidine and derivatives thereof; X represents a hydrogen atom or a hydroxyl group; and R represents a hydrogen atom or a phosphate residue; and a pharmaceutical composition containing any one of the compounds and a pharmaceutically acceptable carrier. Preferably, the composition is used as an anti-HIV agent or a drug for treating AIDS.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to 4′-C-ethynyl nucleosides and the usethereof for producing pharmaceuticals, and more particularly to the usethereof in treating acquired immunodeficiency syndrome (AIDS).

2. Background Art

The clinical setting for AIDS has been dramatically changed by amulti-drug therapy called highly active antiretroviral therapy, orHAART. In this therapy, nucleoside reverse transcriptase inhibitors(NRTIs) such as zidovudine (AZT), didanosine (ddI), zalcitabine (ddC),stavudine (d4T), and lamivudine (3TC) and protease inhibitors (PIs) areemployed in combination. Application of this therapy has drasticallydecreased the number of deaths due to AIDS in many countries (Textbookof AIDS Medicine, p751 (Williams & Wilkins, Baltimore, 1999)).

In spite of the decrease in AIDS-related deaths due to HAART, there hasemerged a multi-drug resistant HIV-1 (human immunodeficiency virus-1)mutant exhibiting cross-resistance to various drugs. For example, in theearly 1990s patients infected with an HIV exhibiting resistance to bothAZT and 3TC were very rare, whereas the percentage of AIDS patientsinfected with such an HIV was as high as 42% in 1995-1996 (AIDS, 11,1184(1997)).

It has been reported that such multi-drug resistant viruses cause 30-60%of drug failure cases in which the viremia level drops once below thedetection limit and then revives to exhibit lasting viremia (AIDS, 12,1631(1998)). Thus, the present status of AIDS treatment is serious.

Conventionally, in terms of a compound which exhibits potent antiviralactivities against multi-drug resistant viruses, there have been knownonly a few protease inhibitors; e.g., JE-2147, which have potentantiviral activity against a multi-PI resistant HIV-1 (Proc. Natl. Acad.Sci. USA, 96,8675(1999)). However, no nucleoside derivative having suchpotent activities has been reported yet.

Ohrui, one of the inventors of the present invention, has synthesized1-(4-C-ethynyl-β-D-ribo-pentofuranosyl)thymine, 4′-C-ethynyluridine, and4′-C-ethynylcytidine and assayed biological activities such as antiviraland antitumor activities thereof. However, no such biological activitieshave been observed for these compounds (Biosci. Biotechnol. Biochem.,63(4), 736-742, 1999).

Furthermore, Matsuda et al. have synthesized 4′-C-ethynylthymidine andassayed the anti-HIV activity thereof. The anti-HIV activity of thecompound is weaker than that of AZT. However, the assay described byMatsuda et al. (Bioorg. Med. Chem. Lett., 9(1999), 385-388) is drawn toan ordinary assay for determining anti-HIV activity on the basis of MT-4cells versus an HIV-1 III_(b) strain, and does not use a multi-drugresistant virus strain.

SUMMARY OF THE INVENTION

In order to find a compound having more potent antiviral activity thanAZT, the present inventors have synthesized a variety of 4′-C-ethynylnucleosides and evaluated the antiviral activity thereof, and have foundthat: 1) a 4′-ethynyl nucleoside derviative having a specific structureexhibits potent anti-HIV activity equal to or greater than that of AZT;2) the compound has potent antiviral activity against a multi-drugresistant virus strain exhibiting resistance to various anti-HIV drugssuch as AZT, ddI, ddC, d4T, and 3TC; and 3) the compound exhibits nosignificant cytotoxicity. The present invention has been accomplished onthe basis of these findings.

Accordingly, the present invention provides 4′-C-ethynyl nucleosides(other than 4′-C-ethynylthymidine) represented by formula [I]:

wherein B represents a base selected from the group consisting ofpyrimidine, purine, and derivatives thereof; X represents a hydrogenatom or a hydroxyl group; and R represents a hydrogen atom or aphosphate residue.

The present invention also provides a pharmaceutical compositioncontaining any one of the compounds and a pharmaceutically acceptablecarrier.

Preferably, the composition is employed as an antiviral drug or a drugfor treating AIDS.

The present invention also provides use, as pharmaceuticals, ofcompounds represented by formula [1].

The present invention also provides a method for treatment of AIDS,comprising administering a compound of formula [1] to a vertebrate,including human.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS (1) Compounds

The compounds of the present invention are represented by formula [I].Examples of bases in formula [I] represented by B include pyrimidines;purines, including azapurines and deazapurines; and derivatives thereof.

Examples of substituents in the bases includes a halogen atom, an alkylgroup, a haloalkyl group, an alkenyl group, a haloalkenyl group, analkynyl group, an amino group, an alkylamino group, a hydroxyl group, ahydroxyamino group, an aminoxy group, an alkoxy group, a mercapto group,an alkylmercapto group, an aryl group, an aryloxy group, and a cyanogroup. The number and substitution site of these substituents are notparticularly limited.

Examples of halogen atoms serving as substituents include chlorine,fluorine, iodine, and bromine. Examples of alkyl groups include C1-C7alkyl group such as methyl, ethyl, and propyl. Examples of haloalkylgroups include C1-C7 haloalkyl groups such as fluoromethyl,difluoromethyl, trifluoromethyl, bromomethyl, and bromoethyl. Examplesof alkenyl groups include C2-C7 alkenyl groups such as vinyl and allyl.Examples of haloalkenyl groups include C2-C7 haloalkenyl groups such asbromovinyl and chlorovinyl. Examples of alkynyl groups include C2-C7alkynyl groups such as ethynyl and propynyl. Examples of alkylaminogroups include C1-C7 alkylamino groups such as methylamino andethylamino.

Examples of alkoxy groups include C1-C7 alkoxy groups such as methoxyand ethoxy. Examples of alkylmercapto groups include C1-C7 alkylmercaptogroups such as methylmercapto and ethylmercapto. Examples of aryl groupsinclude a phenyl group; alkylphenyl groups having a C1-C5 alkyl such asmethylphenyl and ethylphenyl; alkoxyphenyl groups having a C1-C5 alkoxysuch as methoxyphenyl and ethoxyphenyl; alkylaminophenyl groups having aC1-C5 alkyl such as dimethylaminophenyl and diethylaminophenyl; andhalogenophenyl groups such as chlorophenyl and bromophenyl.

Examples of pyrimidine bases and derivatives thereof include cytosine,uracil, 5-fluorocytosine, 5-fluorouracil, 5-chlorocytosine,5-chlorouracil, 5-bromocytosine, 5-bromouracil, 5-iodocytosine,5-iodouracil, 5-methylcytosine, 5-ethylcytosine, 5-methyluracil(thymine), 5-ethyluracil, 5-fluoromethylcytosine, 5-fluorouracil,5-trifluorocytosine, 5-trifluorouracil, 5-vinyluracil,5-bromovinyluracil, 5-chlorovinyluracil, 5-ethynylcytosine,5-ethynyluracil, 5-propynyluracil, pyrimidin-2-one,4-hydroxyaminopyrimidin-2-one, 4-aminoxypyrimidin-2-one,4-methoxypyrimidin-2-one, 4-acetoxypyrimidin-2-one,4-fluoropyrimidin-2-one, and 5-fluoropyrimidin-2-one.

Examples of purine bases and derivatives thereof include purine,6-aminopurine (adenine), 6-hydroxypurine, 6-fluoropurine,6-chloropurine, 6-methylaminopurine, 6-dimethylaminopurine,6-trifluoromethylaminopurine, 6-benzoylaminopurine,6-acethylaminopurine, 6-hydroxyaminopurine, 6-aminoxypurine,6-methoxypurine, 6-acetoxypurine, 6-benzoyloxypurine, 6-methylpurine,6-ethylpurine, 6-trifluoromethylpurine, 6-phenylpurine,6-mercaputopurine, 6-methylmercaputopurine, 6-aminopurine-1-oxide,6-hydroxypurine-1-oxide, 2-amino-6-hydroxypurine (guanine),2,6-diaminopurine, 2-amino-6-chloropurine, 2-amino-6-iodepurine,2-aminopurine, 2-amino-6-mercaptopurine, 2-amino-6-methylmercaptopurine,2-amino-6-hydroxyaminopurine, 2-amino-6-methoxypurine,2-amino-6-benzoyloxypurine, 2-amino-6-acetoxypurine,2-amino-6-methylpurine, 2-amino-6-cyclopropylaminomethylpurine,2-amino-6-phenylpurine, 2-amino-8-bromopurine, 6-cyanopurine,6-amino-2-chloropurine (2-chloroadenine), 6-amino-2-fluoropurine(2-fluoroadenine), 6-amino-3-deazapurine, 6-amino-8-azapurine,2-amino-6-hydroxy-8-azapurine, 6-amino-7-deazapurine,6-amino-1-deazapurine, and 6-amino-2-azapurine.

When B is a pyrimidine base and X is a hydrogen atom, examples ofcompounds represented by formula [I] include the following compounds:

4′-C-ethynyl-2′-deoxycytidine,

4′-C-ethynyl-2′-deoxy-5-halogenocytidine,

4′-C-ethynyl-2′-deoxy-5-alkylcytidine,

4′-C-ethynyl-2′-deoxy-5-haloalkylcytidine,

4′-C-ethynyl-2′-deoxy-5-alkenylcytidine,

4′-C-ethynyl-2′-deoxy-5-haloalkenylcytidine,

4′-C-ethynyl-2′-deoxy-5-alkynylcytidine,

4′-C-ethynyl-2′-deoxy-5-halogenouridine,

4′-C-ethynyl-2′-deoxy-5-alkyluridine (other than 4′-C-ethynylthymidine),

4′-C-ethynyl-2′-deoxy-5-haloalkyluridine,

4′-C-ethynyl-2′-deoxy-5-alkenyluridine,

4′-C-ethynyl-2′-deoxy-5-haloalkenyluridine, and

4′-C-ethynyl-2′-deoxy-5-alkynyluridine, and 5′-phoshate esters thereof.

When B is a pyrimidine base and X is a hydroxyl group, examples ofcompounds represented by formula [I] include the following compounds:

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)cytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-halogenocytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkylcytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-haloalkylcytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkenylcytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-haloalkenylcytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkynylcytosine,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-halogenouracil,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkyluracil,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-haloalkyluracil,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkenyluracil,

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-haloalkenyluracil, and

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-alkynyluracil, and5′-phoshate esters thereof.

When B is a purine base and X is a hydrogen atom, examples of compoundsrepresented by formula [I] include the following compounds:

4′-C-ethynyl-2′-deoxyadenosine,

4′-C-ethynyl-2′-deoxyguanosine,

4′-C-ethynyl-2′-deoxyinosine,

9-(4-C-ethynyl-2-deoxy-β-D-ribo-furanosyl)purine, and

9-(4-C-ethynyl-2-deoxy-β-D-ribo-furanosyl)-2,6-diaminopurine,

and 5′-phoshate esters thereof.

When B is a purine base and X is a hydroxyl group, examples of compoundsrepresented by formula [I] include the following compounds:

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)adenine,

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)guanine,

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)hypoxanthine,

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)purine, and

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-2,6-diaminopurine,

and 5′-phoshate esters thereof.

Examples of preferred compounds of the present invention includes thefollowing compounds:

(i) 4′-C-ethynyl pyrimidine nucleosides including

(1) a compound represented by formula [I] wherein X is a hydrogen atom,

(2) a compound represented by formula [I] wherein X is a hydroxyl group,

(3) a compound represented by formula [I] wherein B is cytosine,

(4) a compound represented by formula [I] wherein B is cytosine and X isa hydrogen atom,

(5) a compound represented by formula [I] wherein B is cytosine and X isa hydroxyl group,

(6) 4′-C-ethynyl-2′-deoxycytidine,

(7) 4′-C-ethynyl-2′-deoxy-5-fluorocytidine, and

(8) 1-(4-C-ethynyl-β-D-arabinofuranosyl)cytosine, and

(ii) 4′-C-ethynyl purine nucleosides including

(1) a compound represented by formula [I] wherein X is a hydrogen atom,

(2) a compound represented by formula [I] wherein X is a hydroxyl group,

(3) a compound represented by formula [I] wherein B is selected from thegroup consisting of adenine, guanine, hypoxanthine, and diaminopurine,

(4) a compound represented by formula [I] wherein B is selected from thegroup consisting of adenine, guanine, hypoxanthine, and diaminopurineand X is a hydrogen atom,

(5) a compound represented by formula [I] wherein B is selected from thegroup consisting of adenine, guanine, hypoxanthine, and diaminopurineand X is a hydroxyl group,

(6) 4′-C-ethynyl-2′-deoxyadenosine,

(7) 4′-C-ethynyl-2′-deoxyguanosine,

(8) 4′-C-ethynyl-2′-deoxyinosine,

(9) 9-(4-C-ethynyl-2-deoxy-β-D-ribo-pentofuranosyl)-2,6-diaminopurine,and

(10) 9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)adenine.

The compounds of the present invention may be salts, hydrates, orsolvates. When R is a hydrogen atom, examples of salts includeacid-adducts such as hydrochlorides and sulfates. When R is a phosphateresidue, examples of salts include alkali metal salts such as sodiumsalts, potassium salts, and lithium salts; alkaline earth metal saltssuch as calcium salts; and ammonium salts. These salts arepharmaceutically acceptable.

Examples of hydrates or solvates include adducts comprising one moleculeof the compound of the present invention or a salt thereof and 0.1-3.0molecules of water or a solvent. In addition, the compounds of thepresent invention encompass a variety of isomers thereof such astautomers.

(2) Method of Production

One of the compounds of the present invention in which X is a hydrogenatom; i.e., a 2′-deoxy derivative, can be produced by the followingsteps.

First Step;

In the first step, a hydroxymethyl group at the 4-position of thecompound represented by [II] is oxidized to thereby form an aldehyde,which is further converted into an alkyne to thereby yield a compoundrepresented by formula

wherein each of R1 and R2 represents a protective group; R3 represents ahydrogen atom or a protective group; and Bn represents a benzyl group.

The starting material of the reaction is a known compound represented byformula [II] (Biosci. Biotech. Biochem., 57, 1433-1438(1993)).

Each of R1 and R2 may be a protective group which is typically employedfor protecting a hydroxyl group. Examples of types of a protectivemoiety containing R1 or R2 include an ether type, an acyl type, a silyltype, and an acetal type. Specific examples protective groups include asilyl group, an acetyl group, a benzyl group, and an isopropylidenylgroup.

When the hydroxymethyl group at the 4-position of the compoundrepresented by [II] is converted into an aldehyde group by use of anoxidizing agent, examples of oxidizing agents include achromium-containing oxidizing agent such as chromicanhydride-pyridine-acetic anhydride composite reagent, pyridiniumchlorochromate, or pyridinium dichromate; a high-valency iodineoxidizing agent such as Dess-Martin reagent; and adimethylsulfoxide-based oxidizing agent such as a combination ofdimethylsulfoxide and any one of acetic anhydride, oxalyl chloride, ordicyclohexyl carbodiimide.

Reaction conditions vary depending on an employed oxidizing agent. Forexample, when oxidation is carried out by use of oxalyl chloride anddimethyl sulfoxide, oxalyl chloride in an amount of 0.5-5 mol anddimethyl sulfoxide in an amount of 1.5-6 mol are added to 1 mol of acompound represented by formula [II] in an organic solvent such asdichloromethane optionally under an inert gas such as argon or nitrogen.The mixture is then allowed to react for approximately 15 minutes to twohours at −100° C. to 0C. Subsequently, a base such as triethylamine isadded in an amount of 2-10 mol to the mixture, and the resultant mixtureis further allowed to react at room temperature for approximately 15minutes to two hours.

The thus-formed aldehyde can be converted into a corresponding alkynethrough carbon-increasing (i.e., C—C bond formation) reaction of thealdehyde; treating the resultant compound with a strong base to therebyform a metal alkynyl compound; and introducing a protective group to themetal alkynyl compound.

Carbon-increasing reaction may be carried out in an organic solvent suchas dichloromethane or dichloroethane, optionally under an inert gas suchas argon or nitrogen. Specifically, 1 mol of the above-produced aldehydeis reacted with 1-5 mol of carbon tetrabromide and 2-10 mol oftriphenylphosphine at 0-50° C. for approximately 15 minutes to threehours.

Treatment with a strong base may be carried out in an organic solventsuch as tetrahydrofuran, 1,4-dioxane, or dimethoxyethane, optionallyunder an inert gas such as argon or nitrogen. Specifically, 1 mol of acompound obtained through carbon-increasing reaction is reacted with 2-4mol of a lithium compound such as n-butyllithium or t-butyllithium at−100° C. to −20° C. for approximately 5-60 minutes.

Furthermore, when a silyl protective group represented by R3 isintroduced into an alkynyl group in the thus-obtained compound, theaforementioned treatment is followed by addition of a silylating agentsuch as chlorotriethylsilane. A protective group can be introduced to ahydroxyl group by use of a customary method. For example, an acetylgroup may be introduced through reaction with an acetylating agent suchas acetic anhydride.

The thus-obtained compound represented by formula [III] may be isolatedand purified through a manner which is employed for isolating andpurifying typical protected saccharides. For example, the crude compoundis partitioned by use of an ethyl acetate-saturated sodium bicarbonatesolution, and the isolated compound is purified by use of a silica gelcolumn.

Second step;

The second step includes condensation of a compound represented byformula [III] and a base represented by B; deoxygenation at the2′-position; removing a protective group of a saccharide portion; andoptionally phosphorylating the hydroxyl group at the 5′-position, tothereby produce a compound represented by formula [I]:

wherein B represents a base selected from the group consisting ofpyrimidine; purine, including azapurine or deazapurine; and a derivativethereof (other than thymine); R represents a hydrogen atom or aphosphate residue; each of R1 and R2 represents a protective group; R3represents a hydrogen atom or a protective group; and Bn represents abenzyl group.

Condensation of a compound represented by formula [III] and a baserepresented by B can be carried out by reacting the compound with thebase in the presence of a Lewis acid.

The base represented by B may be silylated, and silylation may becarried out through a known method. For example, a base is silylated byuse of hexamethylsilazane and trimethylchlorosilane under reflux.

Examples of Lewis acids include trimethylsilyltrifluoromethanesulfonate, tin tetrachloride, zinc chloride, zinciodide, and anhydrous aluminum chloride.

Condensation reaction may be carried out in an organic solvent such asdichloromethane, 1,2-dichloroethane, acetonitrile, or toluene,optionally under an inert gas such as argon or nitrogen. Specifically, 1mol of a compound represented by formula [III] is reacted with 1-10 molof a base represented by B and 0.1-10 mol of Lewis acid at −20° C. to150° C. for approximately 30 minutes to three hours.

Deoxygenation at the 2′-position may be carried out by converting thederivative having a hydroxyl group to the derivative having a group suchas halogen, phenoxythiocarbonyl, thiocarbonylimidazolyl, ormethyldithiocarbonyl and reducing the converted derivative using aradical reducing agent in the presence of a radical initiator.

For example, when deoxygenation is carried out throughphenoxythiocarbonate, conversion of a hydroxyl group to aphenoxythiocarbonyl group may be carried out in an organic solvent, suchas tetrahydrofuran, acetonitrile, or dichloromethane, in the presence ofa base such as dimethylaminopyridine or pyridine, optionally under aninert gas such as argon or nitrogen. Specifically, 1 mol of theaforementioned condensation product in which only the protective groupfor the hydroxyl group at the 2′-position had been eliminated is reactedunder stirring with 1-10 mol, preferably 1.1-2 mol, of a phenylchlorothionoformate derivative at 0-50° C. for approximately 0.5-5hours. Alternatively, when deoxygenation is carried out via a bromocompound, the bromination may be carried out in an organic solvent, suchas tetrahydrofuran, acetonitrile, or dichloromethane, by use of abrominating agent such as acetyl bromide at 0-150° C. for approximately0.5-5 hours, optionally under an inert gas such as argon or nitrogen.The brominating agent is used in an amount of 1-50 mol, preferably 5-20mol, per mol of the aforementioned condensate from which a protectivegroup at the 2′-position had been removed.

Subsequently, reduction may be carried out in an organic solvent such astoluene or benzene in the presence of a radical initiator such asazobisisobutyronitrile, optionally under an inert gas such as argon ornitrogen. Specifically, 1 mol of the aforementioned phenoxythiocarbonateor bromide is reacted under stirring with 1-10 mol, preferably 2-5 mol,of a radical reducing agent such as tributyltin hydride at 50-150° C.for approximately 1-5 hours.

One of the compounds of the present invention in which X is a hydroxylgroup; i.e., an arabino derivative, can be produced by the followingsteps.

First step;

The first step includes condensation of a compound represented byformula [III] and a base represented by B; stereochemically invertingthe hydroxyl group at the 2′-position to be an arabino form; removing aprotective group of a saccharide portion; and optionally phosphorylatingthe hydroxyl group at the 5′-position, to thereby produce a compoundrepresented by formula [I]:

wherein B represents a base selected from the group consisting ofpyrimidine, purine including azapurine or deazapurine, and a derivativethereof; R represents a hydrogen atom or a phosphate residue; each of R1and R2 represents a protective group; R3 represents a hydrogen atom or aprotective group; and Bn represents a benzyl group.

Condensation of a compound represented by formula [III] and a baserepresented by B can be carried out by reacting the compound with thebase in the presence of a Lewis acid.

The base represented by B may be silylated, and silylation may becarried out through a known method. For example, a base is silylated byuse of hexamethylsilazane and trimethylchlorosilane under reflux.

Examples of Lewis acids include trimethylsilyltrifluoromethanesulfonate, tin tetrachloride, zinc chloride, zinciodide, and anhydrous aluminum chloride.

Condensation reaction may be carried out in an organic solvent such asdichloromethane, 1,2-dichloroethane, acetonitrile, or toluene,optionally under an inert gas such as argon or nitrogen. Specifically, 1mol of a compound represented by formula [III] is reacted with 1-10 molof a base represented by B and 0.1-10 mol of Lewis acid at −20° C. to150° C. for approximately 30 minutes to three hours.

Stereo-inversion of the hydroxyl group at the 2′-position can be carriedout by converting a compound containing the hydroxyl into acorresponding 2,2′-anhydrocyclonucleoside and hydrolyzing thenucleoside. Anhydrocyclization may be carried out through treatment witha sulfonating agent such as methanesulfonyl chloride, or throughtreatment with a fluorinating agent such as diethylaminosulfurtrifluoride.

For example, when diethylaminosulfur trifluoride is employed,anhydrocyclization may be carried out in an organic solvent such asdichloromethane or toluene, optionally under an inert gas such as argonor nitrogen. Specifically, 1 mol of the aforementioned condensationproduct in which the protective group for the hydroxyl group at the2′-position was removed is reacted with 1.1-5 mol, preferably 1.5-2 mol,of diethylaminosulfur trifluoride at 0° C. to room temperature forapproximately five minutes to 2 hours. Alternatively, whenmethanesulfonyl chloride is employed, anhydrocyclization may be carriedout in an organic solvent such as pyridine, optionally under an inertgas such as nitrogen. Specifically, 1 mol of the aforementionedcondensation product in which the protective group for the hydroxylgroup at the 2′-position had been eliminated is reacted with 1.1-5 mol,preferably 1.5-2 mol, of methanesulfonyl chloride at 0-50° C. forapproximately five minutes to 10 hours.

Subsequently, hydrolysis may be carried out in the presence of anappropriate base or acid catalyst. For example, when a base catalyst isemployed, hydrolysis may be carried out in a solvent mixture comprisingwater and an alcoholic solvent such as ethanol in the presence of a basesuch as sodium hydroxide or potassium hydroxide at room temperature to100° C. for approximately 30 minutes to 5 hours.

In the case in which a base represented by B in the target compound;i.e., 4′-ethynylnucleoside, is a base having an amino group, the targetcompound may also be produced from a hydroxyl-containing base compoundthrough a known method. For example, if the 4-position of a pyrimidinebase is sought to be aminated, the hydroxyl group at the 4-position of apyrimidine base may be converted into a group such as chloro, silyloxy,alkyloxy, sulfonyloxy, or triazolyl, and then the converted group isreacted with ammonia. For example, amination through a triazolederivative may be carried out with stirring in an organic solvent suchas dichloromethane, acetonitrile, dimethylformamide, or pyridine in thepresence of a base such as triethylamine (triethylamine may be omittedif pyridine is used as a solvent) and a phosphorylating agent such as4-chlorophenylphosphorodichloridate, optionally under an inert gas suchas argon or nitrogen. Specifically, 1 mol of the aforementionedcondensation product is reacted with 1-20 mol, preferably 2-10 mol, of1,2,4-triazole at 0° C. to room temperature for approximately 12-72hours, followed by addition of aqueous ammonia in an appropriate amountand further reaction at 0° C. to room temperature for approximately 1-12hours.

In addition, an amino group in a base may be removed through aconventional method making use of any of a variety of deaminases, suchas adenosine deaminase or cytidine deaminase.

Finally, a protective group of the thus-produced nucleoside is removed,to thereby obtain the compounds (R=H) of the present invention.

A protective group may be removed through a method appropriatelyselected from a routine procedure such as hydrolysis under acidicconditions, hydrolysis under basic conditions, treatment withtetrabutylammonium fluoride, or catalytic reduction, in accordance withthe protective group employed.

When R in a target compound is a phosphate residue such as monophosphateor diphosphate, a compound in which R is a hydrogen atom is reacted witha phosphorylating agent; e.g., phosphorus oxychloride ortetrachloropyrophosphoric acid, which selectively phosphorylates the5′-position of a nucleoside, to thereby produce a target compound in afree or salt form.

The compounds of the present invention may be isolated and purifiedthrough conventional methods, in appropriate combination, which areemployed for isolating and purifying nucleosides and nucleotides; e.g.,recrystallization, ion-exchange column chromatography, and adsorptioncolumn chromatography. The thus-obtained compounds may further beconverted to a salt thereof in accordance with needs.

(3) Use

As shown in the below-described Test Examples, the compounds of thepresent invention exhibit excellent antiviral activity againstherpesvirus or retrovirus. Thus, the compositions of the presentinvention containing one of the compounds of the present invention as anactive ingredient can be used as therapeutic drugs. Specifically, thecompositions of the present invention are useful for the treatment ofinfectious diseases caused by herpesvirus or retrovirus, in particular,AIDS, which is caused by HIV infection.

Examples of target viruses include viruses belonging to Herpesviridaesuch as herpes simplex virus type 1, herpes simplex virus type 2, orvaricella-zoster virus, and Retroviridae such as human immunodeficiencyvirus.

The dose of the compounds of the present invention depends on and isdetermined in consideration of conditions such as the age, body weight,and type of disease of the patient; the severity of a disease of thepatient; the drug tolerance; and the administration route. However, thedose per day and per body weight is selected typically within0.00001-1,000 mg/kg, preferably 0.0001-100 mg/kg. The compounds areadministered in a single or divided manner.

Any administration route may be employed, and the compounds may beadministered orally, parenterally, enterally, or topically.

When a pharmaceutical is prepared from the compounds of the presentinvention, the compounds are typically mixed with customarily employedadditives, such as a carrier and an excipient. Examples of solidcarriers include lactose, kaolin, sucrose, crystalline cellulose, cornstarch, talc, agar, pectin, stearic acid, magnesium stearate, lecitin,and sodium chloride. Examples of liquid carriers include glycerin,peanut oil, polyvinylpyrrolidone, olive oil, ethanol, benzyl alcohol,propylene glycol, and water.

The dosage form is arbitrarily selected. When the carrier is solid,examples of dosage forms include tablets, powder, granules, capsules,suppositories, and troches, whereas when it is liquid, examples includesyrup, emulsion, soft-gelatin-encapsulated, cream, gel, paste, spray,and injection.

As shown in the below-described results of Test Examples, the compoundsof the present invention exhibit excellent anti-HIV activity,particularly against multi-drug resistant HIV strains having resistanceto various of anti-HIV drugs such as AZT, DDI, DDC, D4T, and 3TC. Thecompounds have no significant cytotoxicity. Thus, the compounds of thepresent invention are expected to be developed for producingpharmaceuticals, particularly drugs for treating AIDS.

EXAMPLES

The present invention will next be described in detail by way ofexamples including Synthesis Examples, Test Examples, and DrugPreparation Examples, which should not be construed as limiting theinvention thereto.

Synthesis Example 1

(1) Synthesis of4-C-formyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribo-pentofuranose(Compound 2)

Oxalyl chloride (3.38 ml, 38.7 mmol) was dissolved in dichloromethane(80.0 ml), and dimethylsulfoxide (5.50 ml, 77.5 mmol) was added dropwiseto the solution at −78° C. in an argon atmosphere, followed by stirringfor 15 minutes at the same temperature. A solution (100 ml) of4-C-hydroxymethyl-3,5-di-O-benzyl-1,2-O-isopropylidene-β-D-ribo-pentofuranose(Compound 1) (10.3 g, 25.7 mmol) in dichloromethane was added dropwiseto the solution at −78° C., and the mixture was stirred for 30 minutes.After triethylamine (10.9 ml, 77.6 mmol) was added thereto, the reactionmixture was allowed to warm to room temperature, followed by stirringfor 30 minutes. After water was added to the mixture with stirring, theorganic layer was dried over anhydrous magnesium sulfate and wasconcentrated through distillation under reduced pressure. The residuewas purified by means of silica gel column chromatography (silica gel1500 ml, eluent; n-hexane:ethyl acetate=2:1), to thereby yield acolorless viscous compound (Compound 2; 9.68 g, 24.3 mmol, 94.1%).

¹H-NMR(CDCl₃) δ 9.92 (1H, s, formyl), 7.33-7.24 (10H, m, aromatic), 5.84(1H, d, H-1 J_(1,2)=3.30), 4.71, 4.59 (each 1H, d, benzyl,J_(gem)=12.00), 4.60 (1H, br.t, H-2), 4.52, 4.46 (each 1H, d, benzyl,J_(gem)=12.00), 4.37 (1H, d, H-3, J_(2,3)=4.50), 3.68, 3.61 (each 1H, d,H-5, J_(gem)=10.95), 1.60, 1.35 (each 3H, s, acetonide); EIMS m/z:398(M⁺). HRMS m/z(M⁺): Calcd. for C₂₃H₂₆O₆: 398.1729, Found: 398.1732.[α]_(D) +24.5° (c=1.03, CHCl₃).

(2) Synthesis of 4-C-(2,2-dibromoethenyl)-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribo-pentofuranose (Compound 3)

Compound 2 (9.50 g, 23.8 mmol) was dissolved in dichloromethane (200ml), and carbon tetrabromide (15.8 g, 47.6 mmol) and triphenylphosphine(25.0 g, 95.3 mmol) were added to the solution under ice-cooling,followed by stirring at room temperature for one hour. Triethylamine(20.0 ml, 142 mmol) was added to the mixture, followed by stirring for10 minutes. The reaction mixture was poured into n-hexane (1000 ml) andthe produced precipitates were separated through filtration. Thefiltrate was concentrated through distillation under reduced pressure,and the residue was purified by means of silica gel columnchromatography (silica gel 1500 ml, eluent; n-hexane:ethyl acetate=3:1),to thereby yield a colorless viscous compound (Compound 3; 12.6 g, 22.7mmol, 95.4%).

¹H-NMR(CDCl₃) δ 7.34-7.24 (10H, m, aromatic), 7.16 (1H, s, Br₂C═CH—),5.76 (1H, d, H-1 J_(1,2)=3.90), 4.72, 4.60 (each 1H, d, benzyl,J_(gem)=12.00), 4.53 (1H, br.t, H-2), 4.60, 4.42 (each 1H, d, benzyl,J_(gem)=12.00), 4.21 (1H, d, H-3, J_(2,3)=4.80), 3.83, 3.39 (each 1H, d,H-5, J_(gem)=11.40), 1.59, 1.30 (each 3H, s, acetonide); EIMS m/z: 473,475 (M-Br). [α]_(D) +6.20° (c=1.00, CHCl₃).

(3) Synthesis of4-C-ethynyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribo-pentofuranose(Compound 4)

Compound 3 (12.4 g, 22.4 mmol) was dissolved in dry tetrahydrofuran (160ml), and a 1.6 M n-butyl lithium (30.7 ml, 49.1 mmol) in n-hexane wasadded to the solution at −78° C. in an argon atmosphere, followed bystirring for 30 minutes at the same temperature. After water was addedto the mixture with stirring, the organic layer was dried over anhydrousmagnesium sulfate and concentrated through distillation under reducedpressure. The residue was purified by means of silica gel columnchromatography (silica gel 1500 ml, eluent; n-hexane:ethyl acetate=3:1),to thereby yield a colorless viscous compound (Compound 4; 7.95 g, 20.2mmol, 90.3%).

¹H-NMR(CDCl₃) δ 7.39-7.22 (10H, m, aromatic), 5.70 (1H, d, H-1J_(1,2)=3.60), 4.78, 4.69 (each 1H, d, benzyl, J_(gem)=12.60), 4.55 (1H,br.t, H-2), 4.53, 4.44 (each 1H, d, benzyl, J_(gem)=12.30), 4.16 (1H, d,H-3, J_(2,3)=4.50), 3.71, 3.56 (each 1H, d, H-5, J_(gem)=11.40), 1.73,1.33 (each 3H, s, acetonide); EIMS m/z: 394(M⁺). HRMS m/z(M⁺): Calcd.for C₂₄H₂₆O₅: 394.1780, Found: 394.1777; [α]_(D) +22.60° (c=1.00,CHCl₃).

(4) Synthesis of4-C-triethylsilylethynyl-3,5-di-O-benzyl-1,2-O-isopropylidene-α-D-ribo-pentofuranose(Compound 5)

Compound 4 (5.00 g, 12.7 mmol) was dissolved in dry tetrahydrofuran (100ml), and a 1.6 M n-butyl lithium (9.50 ml, 15.2 mmol) in n-hexane wasadded to the solution at −78° C. in an argon atmosphere, followed bystirring for five minutes at the same temperature. Under the sameconditions, chlorotriethylsilane (2.55 ml, 15.2 mmol) was added thereto,followed by stirring for 30 minutes. After water was added to themixture with stirring, the organic layer was dried over anhydrousmagnesium sulfate and concentrated through distillation under reducedpressure. The residue was purified by means of silica gel columnchromatography (silica gel 1000 ml, eluent; n-hexane:ethyl acetate=3:1),to thereby yield a colorless oily compound (Compound 5; 6.32 g, 12.4mmol, 97.6%).

¹H-NMR(CDCl₃) δ 7.41-7.22 (10H, m, aromatic), 5.71 (1H, d, H-1,J_(1, 2)=3.85), 4.77, 4.65 (each 1H, d, benzyl, J_(gem)=12.09), 4.63(1H, br.t, H-2), 4.57, 4.48 (each 1H, d, benzyl, J_(gem)=12.09), 4.23(1H, d, H-3, J₂, 3=4.67), 1.73, 1.33 (each 3H, s, acetonide), 0.98 (9H,t, Si-CH₂—CH₃, J=7.83), 0.60 (6H, Si—CH₂—CH₃, J=7.97); EIMS m/z:508(M⁺). HRMS m/z(M⁺): Calcd. for C₃₀H₄₀O₅Si: 508, 2645, Found:508,2642; [α]_(D) −27.27° (c=1.045, CHCl₃).

(5) Synthesis of 4-C-triethylsilylethynyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribo-pentofuranose (Compound 6)

Compound 5 (5.55 g, 10.9 mmol) was dissolved in acetic acid (70.0 ml),and trifluoroacetic acid (10.0 ml) and water (30.0 ml) were added to thesolution, followed by stirring overnight at room temperature. Afterdisappearance of Compound 5 had been confirmed by means of silica gelthin-layer chromatography, the reaction mixture was concentrated throughdistillation under reduced pressure. The residue was furtherconcentrated by co-boiling with toluene three times, and then dissolvedin pyridine (50.0 ml). Acetic anhydride (10.3 ml, 0.11 mol) was addedthereto, followed by stirring overnight at room temperature. Thereaction mixture was concentrated through distillation under reducedpressure, and the residue was dissolved in ethyl acetate. The organiclayer was washed with water, dried over anhydrous magnesium sulfate, andconcentrated through distillation under reduced pressure. The residuewas purified by means of silica gel column chromatography (silica gel1000 ml, eluent; n-hexane:ethyl acetate=5:1), to thereby yield acolorless viscous compound (Compound 6; 4.80 g, 8.68 mmol, 79.6%) as ananomer mixture (α:β=1:6.6).

¹H-NMR for α anomer (CDCl₃) δ 7.38-7.28 (10H, m, aromatic), 6.39 (1H, d,H-1, J_(1,2)=4.67), 5.13 (1H, dd, H-2, J_(1,2)=4.67, J_(2, 3)=6.87),4.80, 4.55 (each 1H, benzyl, d, J_(gem)=12.09), 4.61, 4.52 (each 1H, d,benzyl, J_(gem)=12.09), 4.30 (1H, d, H-3, J_(2, 3)=6.87), 3.62 (2H, d,H-5, J=0.55), 2.12, 2.07 (each 3H, s. acetyl), 0.94 (9H, t, Si—CH₂—CH₃,J=7.97), 0.55 (6H, Si—CH₂—CH₃, J=7.97); [α]_(D) −21.8° (c=1.00, CHCl₃).

¹H-NMR for β anomer (CDCl₃) δ 7.35-7.24 (10H, m, aromatic), 6.20 (1H, d,H-1, J_(1,2)=0.82), 5.33 (1H, dd, H-2, J_(1,2)=0.82, J_(2, 3)=4.67),4.66, 4.61 (each 1H, benzyl, d, J_(gem)=11.81), 4.56, 4.47 (each 1H,benzyl, d, J_(gem)=11.81), 4.48 (1H, d, H-3, J_(2, 3)=4.67), 3.69, 3.62(each 1H, d, H-5, J_(gem)=10.99), 2.09, 1.84 (each 3H, s. acetyl), 0.96(9H, t, Si—CH₂—CH₃, J=7.97), 0.58 (6H, Si—CH₂—CH₃, J=7.97) [α]_(D)−58.0° (c=1.00, CHCl₃); EIMS m/z: 552(M⁺). HRMS m/z(M⁺): Calcd. forC₃₁H₄₀O₇Si: 552.2543, Found: 552.2551;

(6) Synthesis of 4′-C-triethylsilylethynyl-2′-O-acetyl-3′,5′-di-O-benzyluridine (Compound 7)

Compound 6 (3.00 g, 5.43 mmol) was dissolved in 1,2-dichloroethane (100ml), and uracil (1.52 g, 13.6 mmol) and N,O-bis(trimethylsilyl)acetamide(9.40 ml, 38.0 mmol) were added to the solution, followed by refluxingfor one hour. After the reaction mixture was allowed to cool to roomtemperature, trimethylsilyl trifluoromethanesulfonate (1.97 ml, 10.9mmol) was added thereto, followed by stirring overnight at 50° C. Asaturated aqueous solution of sodium hydrogencarbonate was added to themixture, and after stirring, precipitate was filtered. The organic layerwas dried over anhydrous magnesium sulfate and concentrated throughdistillation under reduced pressure. The residue was purified by meansof silica gel column chromatography (silica gel 300 ml, eluent;n-hexane:ethyl acetate=1:1), to thereby yield a colorless viscouscompound (Compound 7; 2.50 g, 4.13 mmol, 76.1%).

¹H-NMR(CDCl₃) δ 8.63 (1H, br.s, 3-NH), 7.59 (1H, d, 6-H, J_(5, 6)=8.24),7.41-7.24 (10H, m, aromatic), 6.31 (1H, d, H-1′, J_(1′,2′)=4.95), 5.34(1H, d, H-5, J_(5,6)=8.24), 5.21 (1H, dd, H-2′, J_(1′,2′)=4.95,J_(2′,3′)=6.04), 4.71, 4.58 (each 1H, d, benzyl, J_(gem)=11.81), 4.48(2H, s, benzyl), 4.34 (1H, d, H-3′, J_(2′,3′)=6.04), 3.86, 3.67 (each1H, d, H-5′, J_(gem)=10.50), 2.05 (3H, s, acetyl), 0.97 (9H, t,Si—CH₂—CH₃, J=7.95), 0.60 (6H, Si—CH₂—CH₃, J=7.95). FABMS m/z: 605(MH⁺).HRMS m/z(MH⁺): Calcd. for C₃₃H₄₁N₂O₇Si: 605.2683, Found: 605.2683.[α]_(D) −21.970 (c=1.015, CHCl₃).

(7) Synthesis of 4′-C-triethylsilylethynyl-3′,5′-di-O-benzyluridine(Compound 8)

Compound 7 (2.00 g, 3.3 mmol) was dissolved in methanol (90.0 ml), andtriethylamine (10.0 ml) was added to the solution, followed by stirringfor 48 hours at room temperature. The reaction mixture was concentratedthrough distillation under reduced pressure, and the residue waspurified by means of silica gel column chromatography (silica gel 200ml, eluent; n-hexane:ethyl acetate=1:1), to thereby yield a whitepowdery compound (Compound 8; 1.72g, 3.06 mmol, 92.4%).

¹H-NMR(CDCl₃) δ 8.43 (1H, br.s, 3-NH), 7.55 (1H, d, H-6, J_(5,6)=8.24),7.41-7.25 (10H, m, aromatic), 6.10 (1H, d, H-1′, J_(1′,2′)=5.22), 5.37(1H, dd, H-5, J_(5,6)=8.24), 4.96, 4.66 (each 1H, d, benzyl,J_(gem)=11.54), 4.56, 4.50 (each 1H, d, benzyl, J_(gem)=11.00), 4.21(1H, m, H-2′), 4.17 (1H, d, H-3′, J_(2′,3′)=5.77), 3.87, 3.74 (each 1H,d, H-5′, J_(gem)=10.44), 3.02 (1H, br.d, 2′-OH), 0.97 (9H, t,Si—CH₂—CH₃, J=7.69), 0.60 (6H, Si—CH₂—CH₃, J=7.69). FABMS m/z: 563(MH⁺).HRMS m/z(MH⁺): Calcd. for C₃₁H₃₉N₂O₆Si: 563.2577, Found: 563.2586.[α]_(D) −21.56° (c=1.025, CHCl₃); m.p. 119-120° C.

(8) Synthesis of 4′-C-triethylsilylethynyluridine (Compound 9)

Compound 8 (1.50 g. 2.67 mmol) was dissolved in dichloromethane (75.0ml), and a 1.0 M boron trichloride (26.7 ml, 26.7 mmol) indichloromethane was added to the solution at −78° C. in an argonatmosphere, followed by stirring for three hours at the sametemperature. A mixture of pyridine (10.0 ml) and methanol (20.0 ml) wasadded thereto at −78° C., followed by stirring for ten minutes. Thereaction mixture was concentrated through distillation under reducedpressure, and the residue was partitioned with ethyl acetate and water.The organic layer was dried over anhydrous magnesium sulfate andconcentrated through distillation under reduced pressure. The residuewas purified by means of silica gel column chromatography (silica gel200 ml, eluent; chloroform:methanol=9:1), to thereby yield a whitepowdery compound (Compound 9; 0.95 g, 2.48 mmol, 92.9%).

¹H-NMR(CDCl₃) δ 11.36 (1H, d, 3-NH), 7.81 (1H, d, H-6, J_(5,6)=8.24),5.92 (1H, d, H-1′, J_(1′,2′)=6.32), 5.68 (1H, dd, J_(5,6)=8.24), 5.55(1H, t, 5′-OH), 5.33 (1H, d, 2′-OH), 5.16 (1H, d, 3′-OH), 4.13 (1H, dd,H-2′, J_(1′,2′)=6.32, J_(2′,3′)=5.77), 4.07 (1H, t, H-3′,J_(2′,3′)=5.77), 3.58 (1H, d, H-5′), 0.96 (9H, t, Si—CH₂—CH₃, J=7.97),0.57 (6H, Si—CH₂—CH₃, J=7.97). FABMS m/z: 383(MH⁺). HRMS m/z(MH⁺):Calcd. for C₁₇H₂₇N₂O₆Si: 383, 1638, Found: 383.1645. [α]_(D) −4.50°(c=1.00, CH₃OH) m.p. 183-186° C.

(9) Synthesis of 4′-C-triethylsilylethynyl-3′,5′-di-O-acetyl-2′-deoxyuridine (Compound 11)

Compound 9 (0.80 g, 2.09 mmol) was suspended in acetonitrile (20.0 ml),and a solution (20.0 ml) of acetyl bromide (1.55 ml, 21.0 mmol) inacetonitrile was added dropwise to the suspension at 85° C. over 30minutes, followed by refluxing for one hour. After the reaction mixturewas concentrated through distillation under reduced pressure, theresidue was dissolved in ethyl acetate and the solution was washed witha saturated aqueous solution of sodium hydrogencarbonate and a saturatedaqueous solution of sodium chloride. The organic layer was dried overanhydrous magnesium sulfate and concentrated through distillation underreduced pressure, to thereby yield4′-C-triethylsilylethynyl-3′,5′-di-O-acetyl-2′-bromo-2′-deoxyuridine(Compound 10). After the crude product (Compound 10) was concentrated byco-boiling with toluene three times, the product was dissolved in drytoluene (50.0 ml). Hydrogenated tri(n-butyl)tin (1.08 ml, 4.19 mmol) and2,2′-azobis(isobutyronitrile) (0.01 g) were added to the solution at 85°C., and the mixture was heated under stirring for one hour in an argonatmosphere. After the reaction mixture was concentrated throughdistillation under reduced pressure, the residue was purified by meansof silica gel column chromatography (silica gel 300 ml, eluent;toluene:ethyl acetate), to thereby yield a colorless viscous compound(Compound 11; 0.40 g, 42.6%).

¹H-NMR(CDCl₃) δ 7.49 (1H, d, H-6, J_(5,6)=8.24), 6.34 (1H, t, H-1′,J_(1′,2′)=6.46), 5.77 (1H, dd, H-5, J_(5,6)=8.24), 5.37 (1H, dd, H-3′,J_(2′,3′)=4.95, 7.42), 4.42, 4.37 (each 1H, d, H-5′, J_(gem)=11.81),2.62, 2.32 (each 1H, m, H-2′), 2.13 (6H, s, acetyl), 1.00 (9H, t,Si—CH₂—CH₃, J=7.82), 0.63 (6H, Si—CH₂—CH₃, J=7.82). FABMS m/z: 451(MH⁺).HRMS m/z(MH⁺): Calcd. for C₂₁H₃₁N₂O₇Si: 451.1900, Found: 451.1934.[α]_(D) −11.7° (c=1.04, CHCl₃)

(10) Synthesis of 4′-C-ethynyl-2′-deoxycytidine (Compound 13)

Compound 11 (0.30 g, 0.67 mmol) was dissolved in pyridine (15.0 ml), andp-chlorophenylphosphrodichloridate (0.33 ml, 2.00 mmol) was added to thesolution under ice-cooling, followed by stirring for two minutes.1,2,4-Triazole (0.46 g, 6.66 mmol) was added to the mixture, followed bystirring for seven days at room temperature. After disappearance of rawmaterial had been confirmed by means of silica gel thin-layerchromatography, the reaction mixture was concentrated throughdistillation under reduced pressure, and the residue was partitionedwith ethyl acetate and water. The organic layer was dried over anhydrousmagnesium sulfate and concentrated through distillation under reducedpressure. The residue was purified by means of silica gel columnchromatography (silica gel 50 ml, eluent; n-hexane:ethyl acetate=1:3),to thereby yield colorless viscous Compound 12:4-(1,2,4-triazolo)-4′-C-ethynyl-2′-deoxyuridine. Compound 12 wasdissolved in dioxane (30.0 ml), and 25% aqueous ammonia (10.0 ml) wasadded to the solution, followed by stirring overnight at roomtemperature. After disappearance of Compound 12 had been confirmed bymeans of silica gel thin-layer chromatography, the reaction mixture wasconcentrated through distillation under reduced pressure. The residuewas dissolved in methanol (45.0 ml), and an aqueous 1 N solution ofsodium hydroxide (5.00 ml, 5.00 mmol) was added thereto, followed bystirring for two hours at room temperature. Acetic acid (0.29 ml, 5.00mmol) was added to the mixture, and the reaction mixture wasconcentrated through distillation under reduced pressure. The residuewas purified by means of reversed-phase medium-pressure columnchromatography (Wakosil 40C18 50 g, eluent; a 5% aqueous solution ofacetonitrile). The fractions containing Compound 13 were brought todryness under reduced pressure, and the residue was crystallized frommethanol-ether, to thereby yield a white crystalline compound (Compound13; 0.12 g, 0.48 mmol, 71.6%).

¹-H-NMR(DMSO-d₆) δ 7.78 (1H, d, H-6, J_(5,6)=7.50), 7.17 (2H, br.d,NH₂),6.14 (1H, dd, H-1′, J_(1′,2′)=4.76, 7.20), 5.72 (1H, d, H-5,J_(5,6)=7.50), 5.49 (1H, d, 3′-OH), 5.42 (1H, t, 5′-OH), 4.30 (1H, t,H-3′, J_(2′,3′)=7.20), 3.64, 3.58 (each 1H, m, H-5′), 3.48 (1H, s,ethynyl), 2, 25, 2.07 (each 1H, m, H-2′); [α]_(D) +75.00 (c=1.00,CH₃OH); FABMS m/z: 252(MH⁺). HRMS m/z(MH⁺): Calcd. for C₁₁H₁₄N₃O₄:252.0984, Found: 252.0979. UV λ max (CH₃OH) nm (ε): 271 (9227); m.p.220° C. (Dec).

Synthesis Example 2

5-Fluorouracil, 5-ethyluracil, 5-bromovinyluracil, and 5-ethynyluracilwere employed instead of uracil used in Synthesis Example 1 (6), and thereactions were carried out in the same manner as described above (ifnecessary, amination reaction by use of triazole described in (10) wasomitted), to thereby synthesize the following compounds:4′-C-ethynyl-2′-deoxy-5-fluorouridine;

4′-C-ethynyl-2′-deoxy-5-ethyluridine;

4′-C-ethynyl-2′-deoxy-5-bromovinyluridine;

4′-C-ethynyl-2′-deoxy-5-ethynyluridine;

4′-C-ethynyl-2′-deoxy-5-ethylcytidine;

4′-C-ethynyl-2′-deoxy-5-bromovinylcytidine; and

4′-C-ethynyl-2′-deoxy-5-ethynylcytidine.

Synthesis Example 3

(1) Synthesis of4-C-ethynyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribo-pentofuranose(Compound 14)

Compound 4 (6.00 g, 15.2 mmol) was dissolved in acetic acid (70.0 ml),and trifluoroacetic acid (10.0 ml) and water (30.0 ml) were added to thesolution, followed by stirring overnight at room temperature. Afterdisappearance of Compound 4 had been confirmed by means of silica gelthin-layer chromatography, the reaction mixture was concentrated throughdistillation under reduced pressure. The residue was concentrated byco-boiling with toluene three times. The treated residue was dissolvedin pyridine (50.0 ml). Acetic anhydride (14.3 ml, 0.15 mol) was addedthereto, followed by stirring overnight at room temperature. Thereaction mixture was concentrated through distillation under reducedpressure, and the residue was dissolved in ethyl acetate. The organiclayer was washed with water, dried over anhydrous magnesium sulfate, andconcentrated through distillation under reduced pressure. The residuewas purified by means of silica gel column chromatography (silica gel1000 ml, eluent; n-hexane ethyl acetate=2:1), to thereby yield acolorless viscous compound (Compound 14; 5.40 g, 12.3 mmol, 80.9%) as ananomer mixture (α:β=1:3.0).

¹H-NMR for a anomer (CDCl₃) δ 7.39-7.25 (10H, m, aromatic), 6.42 (1H, d,H-1, J_(1,2)=4.67), 5.13 (1H, dd, H-2, J_(1,2)=4.67, J_(2,3)=6.87),4.81, 4.60 (each 1H, benzyl, d, J_(gem)=12.09), 4.59, 4.51 (each 1H, d,benzyl, J_(gem)=12.09), 4.30 (1H, d, H-3, J_(2,3)=6.87), 3.63 (2H, d,H-5, J=0.55), 2.73 (1H, s, ethynyl), 2.10, 2.02 (each 3H, s. acetyl).

¹H-NMR for β anomer (CDCl₃) δ 7.35-7.20 (10H, m, aromatic), 6.21 (1H, d,H-1, J_(1,2)=0.82), 5.40 (1H, dd, H-2, J_(1,2)=0.82, J_(2,3)=4.67),4.66, 4.60 (each 1H, benzyl, d, J_(gem)=11.81), 4.50, 4.47 (each 1H,benzyl, d, J_(gem)=11.81), 4.42 (1H, d, H-3, J_(2,3)=4.67), 3.70, 3.66(each 1H, d, H-5, J_(gem)=10.99), 2.80 (1H, s, ethynyl), 2.08, 1.81(each 3H, s. acetyl). EIMS m/z: 438(M⁺). HRMS m/z(M⁺): Calcd. forC₂₅H₂₆O₇: 438.1679, Found: 438.1681

(2) Synthesis of 4′-C-ethynyl-2′-O-acetyl-3′,5′-di-O-benzyluridine(Compound 15)

Compound 14 (2.50 g, 5.70 mmol) was dissolved in 1,2-dichloroethane(80.0 ml), and uracil (1.60 g, 14.27 mmol) andN,O-bis(trimethylsilyl)acetamide (9.86 ml, 39.74 mmol) were added to thesolution, followed by refluxing for one hour. After the reaction mixturewas allowed to cool to room temperature, trimethylsilyltrifluoromethanesulfonate (2.06 ml, 11.40 mmol) was added thereto,followed by stirring overnight at 50° C. A saturated aqueous solution ofsodium hydrogencarbonate was added to the mixture, and after stirring,precipitate was filtered. The organic layer was dried over anhydrousmagnesium sulfate and concentrated through distillation under reducedpressure. The residue was purified by means of silica gel columnchromatography (silica gel 300 ml, eluent; n-hexane:ethyl acetate=2:3),to thereby yield a colorless viscous compound (Compound 15; 2.44 g, 4.97mmol, 87.2%).

¹H-NMR(CDCl₃) δ 8.52 (1H, br. s, 3-NH), 7.55 (1H, d, 6-H, J_(5,6)=8.24),7.40-7.22 (10H, m, aromatic), 6.25 (1H, d, H-1′, J_(1′,2′)=4.40), 5.33(1H, d, H-5, J_(5,6)=8.24), 5.22 (1H, dd, H-2′, J_(1′,2′)=4.40,J_(2′,3′)=5,77), 4.63 (2H, s, benzyl), 4.45, 4.40 (each 1H, d, benzyl,J_(gem)=10.99), 4.34 (1H, d, H-3′, J_(2′,3′)=5.77), 3.84, 3.62 (each 1H,d, H-5′, J_(gem)=10.58), 2.69 (1H, s, ethynyl), 2.11 (3H, s, acetyl).FABMS m/z: 491(MH⁺). HRMS m/z(MH⁺): Calcd. for C₂₇H₂₇N₂O₇: 491.1818,Found: 491.1821. [α]_(D) 29.0° (c=1.00, CHCl₃).

(3) Synthesis of1-(4-C-ethynyl-2-O-acetyl-3,5-di-O-benzyl-β-D-arabino-pentofuranosyl)uracil(Compound 16)

Compound 15 (2.30 g, 4.69 mmol) was dissolved in methanol (90.0 ml), anda 1 N aqueous solution of sodium hydroxide (10.0 ml) was added to thesolution, followed by stirring for two hours at room temperature. Thereaction mixture was neutralized with acetic acid and then brought todryness under reduced pressure. The residue was dissolved in ethylacetate. The organic layer was washed with water, dried over anhydrousmagnesium sulfate, and brought to dryness under reduced pressure. Theresidue was concentrated by co-boiling with a small amount of pyridinethree times. The product was dissolved in pyridine (50.0 ml), andmethanesulfonyl chloride (0.73 ml, 9.41 mmol) was added to the solutionunder cooling, followed by stirring for three hours. A small amount ofwater was added to the reaction mixture, and the mixture was brought todryness under reduced pressure. The residue was dissolved in ethylacetate, followed by washing with water. The organic layer was driedover anhydrous magnesium sulfate and then brought to dryness underreduced pressure. The residue was dissolved in tetrahydrofuran (30.0ml), and a 1 N aqueous solution of sodium hydroxide (50.0 ml) was addedto the solution, followed by refluxing for one hour. After the reactionmixture was neutralized with acetic acid, the target compound was takenup through extraction with ethyl acetate. The organic layers werecombined and dried over anhydrous magnesium sulfate. The organic layerwas brought to dryness under reduced pressure, and the residue waspurified by means of silica gel column chromatography (silica gel 250ml, eluent; n-hexane:ethyl acetate=1:2), to thereby yield a white powdercompound (Compound 16; 1.54 g, 3.43 mmol, 73.1%).

¹H-NMR(CDCl₃) δ 9.82 (1H, br.s, 3-NH), 7.73 (1H, d, 6-H, J_(5,6)=8.06),7.41-7.19 (10H, m, aromatic), 6.24 (1H, d, H-1′, J_(1′,2′)=5,86), 5.25(1H, d, H-5, J_(5,6)=8.06), 4.88, 4.76 (each 1H, d, benzyl,J_(gem)=12.21), 4.78 (1H, H-2′), 4.52 (1H, 2′-OH), 4.46, 4.39 (each 1H,d, benzyl, J_(gem)=11.11), 4.19 (1H, d, H-3′, J_(2′,3′)=6.59), 3.834,3.64 (each 1H, d, H-5′, J_(gem)=10.62), 2.67 (1H, s, ethynyl). FABMSm/z: 449(MH⁺). HRMS m/z(MH⁺): Calcd. for C₂₅H₂₅N₂O₆: 449.1712, Found:449.1713. [α]_(D) 40.70 (c=1.00, CHCl₃). m.p. 105-106° C.

(4) Synthesis of1-(4-C-ethynyl-2,3,5-tri-O-acetyl-β-D-arabino-pentofuranosyl)uracil(Compound 17)

Compound 16 (1.40 g, 3.12 mmol) was dissolved in dichloromethane (40.0ml), and 1.0 M boron tribromide (15.6 ml, 15.6 mmol) in dichloromethanewas added to the solution at −78° C. in an argon atmosphere, followed bystirring for three hours at the same temperature. A mixture of pyridine(5.00 ml) and methanol (10.0 ml) was added thereto at −78° C., and afterstirring for ten minutes, the reaction mixture was concentrated throughdistillation under reduced pressure. After the residue was concentratedby co-boiling with a small amount of methanol three times and by anotherco-boiling with a small amount of pyridine three times, the residue wasdissolved in pyridine (50.0 ml), and acetic anhydride (4.42 ml, 46.7mmol) was added to the solution, followed by stirring overnight at roomtemperature. The reaction mixture was brought to dryness under reducedpressure, and the residue was concentrated by co-boiling with a smallamount of toluene three times and then partitioned with ethyl acetateand water. The organic layer was dried over anhydrous magnesium sulfateand concentrated through distillation under reduced pressure. Theresidue was purified by means of silica gel column chromatography(silica gel 150 ml, eluent; chloroform:methanol=20:1), to thereby yielda white powdery compound (Compound 17; 1.15g, 2.92 mmol, 93.6%).

¹H-NMR(CDCl₃) δ 8.99 (1H, br. s, 3-NH), 7.42 (1H, d, 6-H,J_(5, 6)=8.24), 6.45 (1H, d, H-1′, J_(1′,2′)=4.95), 5.76 (1H, dd, H-5,J_(5,6)=8.24), 5.55 (1H, dd, H-2′, J_(1′,2′)=4.95, J_(2′,3′)=3.57), 5.34(1H, d, H-3′, J_(2′,3′)=3.57), 4.51, 4.42 (each 1H, d, H-5′,J_(gem)=11.81), 2.73 (1H, s, ethynyl). FABMS m/z: 395(MH⁺). HRMSm/z(MH⁺): Calcd. for C₁₇H₁₉N₂O₉: 395.1090, Found: 395.1092. [α]_(D)18.2° (c=1.00, CHCl₃). m.p. 160-162° C.

(5) Synthesis of 1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)cytosine(Compound 19)

Compound 17 (1.00 g, 2.54 mmol) was dossolved in pyridine (50.0 ml), andp-chlorophenylphosphrodichloridate (1.05 ml, 6.38 mmol) was added to thesolution under ice-cooling, followed by stirring for five minutes.1,2,4-Triazole (1.75 g, 25.3 mmol) was added to the mixture, followed bystirring for seven days at room temperature. After disappearance of rawmaterial had been confirmed by means of silica gel thin-layerchromatography, the reaction mixture was concentrated throughdistillation under reduced pressure, and the residue was partitionedwith ethyl acetate and water. The organic layer was dried over anhydrousmagnesium sulfate and concentrated through distillation under reducedpressure. The residue was purified by means of silica gel columnchromatography (silica gel 50 ml, eluent; n-hexane:ethyl acetate=1:3),to thereby yield a colorless viscous compound (Compound 18:1-(4-C-ethynyl-2,3,5-tri-O-acetyl-β-D-arabino-pentofuranosyl)-4-(1,2,4-triazolo)uracil.Compound 18 was dissolved in dioxane (60.0 ml), and a 25% aqueoussolution of ammonia (20.0 ml) was added to the solution, followed bystirring overnight at room temperature. After disappearance of Compound18 had been confirmed by means of silica gel thin-layer chromatography,the reaction mixture was concentrated through distillation under reducedpressure. The residue was purified by means of reversed-phasemedium-pressure column chromatography (Wakosil 40C18 50 g, eluent; a 3%aqueous solution of acetonitrile). The fractions containing Compound 19were brought to dryness under reduced pressure, and the residue wasdissolved in methanol-ether and crystallized from the same medium, tothereby yield a white crystalline compound (Compound 19; 0.51 g, 1.91mmol, 75.2%).

¹H-NMR(DMSO-d₆) δ 7.52 (1H, d, H-6, J_(5,6)=7.42), 7.10 (2H, br. d,NH₂), 6.17 (1H, dd, H-1′, J_(1′,2′)=6.04), 5.66 (1H, d, H-5,J_(5,6)=7.42), 5.62, 5.49 (each 1H, d, 2′-OH, 3′-OH), 5.42 (1H, t,5′-OH), 4.16 (1H, q, H-2′, J_(1′,2′)=J_(2′,3′)=6.04), 3.97 (1H, t, H-3′,J_(2′,3′)=6.04), 3.58 (2H, m, H-5′), 3.48 (1H, s, ethynyl). [α]_(D)+95.7° (c=1.00, CH₃OH); FABMS m/z: 268(MH⁺). HRMS m/z(MH⁺): Calcd. forC₁₁H₁₄N₃O₅: 268.0933, Found: 268.0965.

UV λ_(max) (CH₃OH) nm (ε): 271 (9350); m.p. ˜′200° C. (December).

Synthesis Example 4

5-Fluorouracil, 5-ethyluracil, 5-bromovinyluracil, and 5-ethynyluracilwere employed instead of uracil used in Synthesis Example 3 (2), and thereactions were carried out in the same manner as described above (ifnecessary, amination reaction by use of triazole described in (5) wasomitted), to thereby synthesize the following compounds:

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-fluorouracil;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-ethyluracil;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-bromovinyluracil;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-ethynyluracil;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-fluorocytosine;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-ethylcytosine;

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-bromovinylcytosine; and

1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-5-ethynylcytosine.

Synthesis Example 5

(1) Synthesis of 2′-O-acetyl-3′,5′-di-O-benzyl-4′-C-triethylsilylethynyladenosine (Compound 20)

To a solution of Compound 6 (1.1 g, 2 mmol) in 1,2-dichloroethane (16.5ml), adenine (0.405 g, 3 mmol) and N,O-bis(trimethylsilyl)acetamide (2.7ml, 11 mmol) were added, followed by refluxing for 1.5 hours. After themixture was allowed to cool to room temperature, trimethylsilyltrifluoromethanesulfonate (0.77 ml, 4 mmol) was added dropwise to themixture under stirring at 0° C. in an argon atmosphere. The mixture wasstirred for 15 minutes at room temperature, refluxed for 24 hours, andallowed to cool to room temperature. A saturated aqueous solution ofsodium hydrogencarbonate was added thereto at 0° C., followed bystirring for 15 minutes at room temperature. Insoluble materials wereremoved through filtration by use of Celite, and then the organic layerwas separated from the filtrate. After an aqueous layer was extractedwith chloroform, the organic layer was washed once with a saturatedaqueous solution of sodium hydrogencarbonate, dried over anhydroussodium sulfate, and concentrated through distillation under reducedpressure, so as to evaporate the solvent. The residue was applied to asilica gel column (15 g, eluent; ethylacetate:n-hexane:ethanol=20:20:1), to thereby yield Compound 20 in anamount of 0.69 g (55%).

¹H-NMR(CDCl₃) δ 8.32 (1H, s, purine-H), 8.01 (1H, s, purine-H),7.27-7.37 (10H, m, 2×Ph), 6.37 (1H, d, J=5.1 Hz, H-1′), 5.60 (1H, t,J=5.6 Hz, H-2′), 5.59 (2H, br s, NH₂), 4.75 (1H, d, J=11.0 Hz, CHH′Ph),4.69 (1H, d, J=5.6 Hz, H-3′), 4.60 (1H, d, J=11.0 Hz, CHEPh), 4.58 (1H,d, J=11.2 Hz, CHH′Ph), 4.51 (1H, d, J=11.0 Hz, CHH′Ph), 3.84 (1H, d,J=11.1 Hz, H-5′), 3.69 (1H, d, J=11.1 Hz, H-5′) 2.03 (3H, s, Ac), 0.98(9H, t, J=8.7 Hz, 3×CH₃CH₂), 0.61 (6H, q, J=8.7 Hz, 3×CH₃C₂).

(2) Synthesis of 3′,5′-di-O-benzyl-4′-C-triethylsilylethynyladenosine(Compound 21)

To a solution of Compound 20 (0.354 g, 0.565 mmol) in methanol (14 ml),triethylamine (3.3 ml) was added, and the mixture was stirred for oneday at room temperature under air-tight condition. The mixture wasconcentrated under reduced pressure. The residue was applied to a silicagel column (10 g, eluent; ethyl acetate:n-hexane:ethanol=20:10:1), tothereby yield Compound 21 in an amount of 0.283 g (86%).

¹H-NMR(CDCl₃) δ 8.30 (1H, s, purine-H), 8.00 (1H, s, purine-H),7.30-7.42 (10H, m, 2×Ph), 6.17 (1H, d, J=5.6 Hz, H-1′), 5.55 (2H, br s,NH₂), 4.97 (1H, d, J=11.1 Hz, CHH′Ph), 4.75-4.80 (1H, m, H-2′), 4.72(1H, d, J=11.1 Hz, CHH′Ph), 4.59 (1H, d, J=11.6 Hz, CHH′Ph), 4.54 (1H,d, J=11.6 Hz, CHH′Ph), 4.50 (1H, d, J=5.6 Hz, H-3′), 3.84 (1H, d, J=11.1Hz, H-5′), 3.74 (1H, d, J=11.1 Hz, H-5′), 3.50 (1H, d, J=8.3 Hz, OH),0.98 (9H, t, J=7.9 Hz, 3×CH₃CH₂), 0.62 (6H, q, J=7.9 Hz, 3×CH₃C₂).

(3) Synthesis of3′,5′-di-O-benzyl-2′-deoxy-4′-C-triethylsilylethynyladenosine (Compound22)

To a solution of Compound 21 (0.18 g, 0.308 mmol) and DMAP (0.113 g,0.924 mmol) in acetonitrile (10.6 ml), 4-fluorophenylchlorothionoformate(0.065 ml, 0.462 mmol) was added dropwise under stirring at roomtemperature in an argon atmosphere and stirred for an hour at roomtemperature, followed by condensation under reduced pressure. Water wasadded to the residue, and the mixture was extracted with ethyl acetate.The organic layer was washed with water and was washed with a saturatedaqueous solution of sodium chloride, dried over anhydrous sodiumsulfate, and the solvent was distilled off under reduced pressure. Theresidue was applied to a silica gel column (eluent; ethylacetate:n-hexane:ethanol=20:20:1), to thereby yield crude thiocarbonate.

The thiocarbonate was dissolved in toluene (9 ml), and hydrogenatedtributyltin (0.41 ml, 1.85 mmol) and 2,2′-azobis(isobutyronitrile)(0.013 g, 0.077 mmol) were added to the solution. The reaction mixturewas stirred at 85° C. for an hour in an argon atmosphere and allowed tocool to room temperature. The solvent was evaporated under reducedpressure. The residue was applied to a silica gel column (20 g, eluent;ethyl acetate:n-hexane:ethanol=20:10:1), to thereby yield Compound 22 inan amount of 0.10 g (57%).

¹H-NMR(CDCl₃) δ 8.32 (1H, s, purine-H), 8.11 (1H, s, purine-H),7.26-7.37 (10H, m, 2×Ph), 6.51 (1H, t, J=6.0 Hz, H-1′), 5.54 (2H, br s,NH₂), 4.72 (1H, d, J=12.0 Hz, CH′Ph), 4.61 (2H, d, J=10.5 Hz, CH₂Ph),4.60 (1H, t, J=6.6 Hz, H-3′), 4.55 (1H, d, J=12.0 Hz, CHH′Ph), 3.88 (1H,d, J=10.7 Hz, H-5′), 3.76 (1H, d, J=10.7 Hz, H-5′), 2.71-2.76 (2H, m,H-2′), 0.99 (9H, t, J=7.8 Hz, 3×CH₃CH₂), 0.62 (6H, q, J=7.5 Hz,3×CH₃CH₂).

(4) Synthesis of 2′-deoxy-4′-C-ethynyladenosine (Compound 23) and9-(2-deoxy-4-C-ethynyl-β-D-ribofuranosyl)purine (Compound 24)

To a solution of Compound 22 (0.23 g, 0.404 mmol) in tetrahydrofuran(9.4 ml), a 1.0 M solution of tetrabutylammonium fluoride (0.44 ml, 0.44mmol) was added under stirring at room temperature, and after stirringfor 30 minutes at the same temperature, the solvent was evaporated underreduced pressure. The residue was applied to a silica gel column andeluted with ethyl acetate, to thereby yield 0.186 g of a crude compoundwith no triethylsilyl group.

A solution of the above-described compound with no triethylsilyl groupin tetrahydrofuran (1.8 ml) and anhydrous ethanol (0.18 ml) were fed toa flask. Ammonia gas was condensed at −78° C. to 18 ml and fed to theflask. Metallic sodium (0.047 g, 2.02 mmol) were added quickly in anargon atmosphere, followed by stirring for 15 minutes at the sametemperature. In addition, metallic sodium (0.023 g) was added to themixture, and after stirring for 10 minutes, ammonium chloride was added.After the mixture was stirred for 1.5 hours at room temperature, ethanolwas added thereto. Insoluble materials were separated through Celite,and washed with ethanol two times. The resultant filtrate and thewashing liquid were concentrated under reduced pressure. The residue wasapplied to a silica gel column (10 g, eluent; ethylacetate:methanol=20:1), to thereby yield a mixture of Compound 23 andCompound 24 in an amount of 0.079 g. Subsequently, the mixture wasapplied to a reversed-phase ODS silica gel column and eluted with a 5%aqueous solution of ethanol, to thereby yield Compound 24 in an amountof 0.028 g (27%), and further eluted with a 7.5% aqueous solution ofethanol, to thereby yield Compound 23 in an amount of 0.021 g (19%).

(Compound 23)

¹H-NMR(DMSO-d₆) δ 8.33 (1H, s, purine-H), 8.15 (1H, s, purine-H), 7.30(2H, br s, NH₂), 6.36 (1H, t, J=6.4 Hz, H-1′), 5.54 (1H, d, J=5.4 Hz,OH), 5.53 (1H, t, J=5.4 Hz, OH), 4.58 (1H, q, J=5.9 Hz, H-3′), 3.66 (1H,dd, J=12.2, 5.4 Hz, H-5′), 3.56 (1H, dd, J=11.7, 7.3 Hz, H-5′), 3.50(1H, s, ethynyl-H), 2.76 (1H, dt, J=13.2, 6.4 Hz, H-2′), 2.41 (1H, dt,J=13.2, 6.8 Hz, H-2′).

(Compound 24)

¹H-NMR(DMSO-d₆) δ 9.18 (1H, s, purine-H), 8.96 (1H, s, purine-H), 8.79(1H, s, purine-H), 6.50 (1H, t, J=7.3, 4.9 Hz, H-1′), 5.60 (1H, d, J=5.9Hz, OH), 5.29 (1H, t, J=5.4 Hz, OH), 4.67 (1H, q, J=5.9 Hz, H-3′), 3.67(1H, dd, J=11.7, 5.9 Hz, H-5′), 3.58 (1H, dd, J=11.7, 6.8 Hz, H-5′),3.53 (1H, s, ethynyl-H), 2.85 (1H, ddd, J=13.2, 6.8, 4.9 Hz, H-2′),2.48-2.56 (1H, m, H-2′).

Synthesis Example 6

(1) Synthesis of9-(2-O-acetyl-3,5-di-O-benzyl-4-C-triethylsilylethynyl-β-D-ribofuranosyl)-2,6-diaminopurine

(Compound 25)

To a solution of Compound 6 (1.1 g, 2 mmol) in 1,2-dichloroethane (16.5ml), diaminopurine (0.45 g, 3 mmol) and N,O-bis(trimethylsilyl)acetamide(4.4 ml, 18 mmol) were added, followed by refluxing for three hours.After the mixture was cooled to room temperature, trimethylsilyltrifluoromethanesulfonate (0.77 ml, 4 mmol) was added dropwise to themixture at 0° C. in an argon atmosphere. The mixture was stirred for 15minutes at room temperature, refluxed for 24 hours, and cooled to roomtemperature. A saturated aqueous solution of sodium hydrogencarbonatewas added thereto at 0° C., followed by stirring for 15 minutes at roomtemperature. Insoluble materials were separated through filtration byuse of Celite, and then the organic layer was separated from thefiltrate. After an aqueous layer was extracted with chloroform once, theorganic layer was washed with a saturated aqueous solution of sodiumchloride, dried over anhydrous sodium sulfate, and the solvent wasevaporated under reduced pressure. The residue was applied to a silicagel column (20 g, eluent; ethyl acetate:n-hexane:ethanol=20:10:1), tothereby yield Compound 25 in an amount of 0.85 g (66%).

¹H-NMR (CDCl₃) δ 7.68 (1H, s, H-8), 7.26-7.37 (10H, m, 2×Ph), 6.17 (1H,d, J=6.5 Hz, H-1′), 5.78 (1H, dd, J=6.5, 6.0 Hz, H-2′), 5.34 (2H, br s,NH₂), 4.76 (1H, d, J=11.4 Hz, CHH′Ph), 4.69 (1H, d, J=6.0 Hz, H-3′),4.61 (1H, d, J=11.4 Hz, CHH′Ph), 4.60 (1H, d, J=11.9 Hz, CHH′Ph), 4.55(2H, br s, NH₂), 4.52 (1H, d, J=11.9 Hz, CHH′Ph), 3.83 (1H, d, J=10.7Hz, H-5′), 3.70 (1H, d, J=10.7 Hz, H-5′), 2.04 (3H, s, Ac), 0.99 (9H, t,J=8.3 Hz, 3×CH₂CH₂), 0.61 (6H, q, J=8.3 Hz, 3×CH₃CH₂).

(2) Synthesis of2,6-diamino-9-(3,5-di-O-benzyl-4-C-triethylsilylethynyl-β-D-ribofuranosyl)purine(Compound 26)

Compound 25 (0.85 g, 1.32 mmol) was treated in the same manner as in thesynthesis of Compound 21, and the resultant residue was applied to asilica gel column (15 g, eluent; ethylacetate:n-hexane:ethanol=30:10:1), to thereby yield Compound 26 in anamount of 0.74 g (93%).

¹H-NMR(CDCl₃) δ 7.70 (1H, s, H-8), 7.29-7.42 (10H, m, 2×Ph), 6.00 (1H,d, J=4.9 Hz, H-1′), 5.35 (2H, br s, NH₂), 4.93 (1H, d, J=11.5 Hz,CHH′Ph),4.74 (1H, d, J=11.5 Hz, CHH′Ph), 4.73 (1H, t, J=5.8 Hz, H-2′),4.60 (1H, d, J=12.0 Hz, CHH′Ph), 4.55 (2H, br s, NH₂), 4.54 (1H, d,J=12.0 Hz, CHH′Ph), 4.49 (1H, d, J=5.9 Hz, H-3′), 3.81 (1H, d, J=10.7Hz, H-5′), 3.72 (1H, d, J=10.7 Hz, H-5′), 3.62 (1H, br s, OH), 0.99 (9H,t, J=7.8 Hz, 3×CH₃CH₂), 0.62 (6H, q, J=7.8 Hz, 3×CH₃CH₂).

(3) Synthesis of2,6-diamino-9-(3,5-di-O-benzyl-2-deoxy-4-C-triethylsilylethynyl-β-D-ribofuranosyl)purine(Compound 27)

Compound 26 (0.103 g, 0.171 mmol) was treated in the same manner as inthe synthesis of Compound 22, and the resultant residue was applied to asilica gel column (10 g, eluent; ethyl acetate:n-hexane :ethanol=30:10:1), to thereby yield Compound 27 in an amount of 0.055 g (55%).

¹H-NMR(CDCl₃) δ 7.79 (1H, s, H-8), 7.26-7.37 (10H, m, 2×Ph), 6.34 (1H,dd, J=6.6, 5.5 Hz, H-1′), 5.36 (2H, br s, NH₂), 4.72 (1H, d, J=11.7 Hz,CHH′Ph), 4.56-4.63 (5H, m, CH₂ Ph, H-3′), 4.57 (1H, d, J=11.7 Hz,CHH′Ph), 3.85 (1H, d, J=10.1 Hz, H-5′), 3.75 (1H, d, J=10.6 Hz, H-5′),2.62-2.73 (2H, m, H-2′), 0.99 (9H, t, J=7.9 Hz, 3×CH₃CH₂), 0.62 (6H, q,J=7.9 Hz, 3×CH₃ CH₂).

(4) Synthesis of2,6-diamino-9-(2-deoxy-4-C-ethynyl-β-D-ribofuranosyl)purine (Compound28)

To a solution of Compound 27 (0.263 g, 0.45 mmol) in tetrahydrofuran(10.3 ml), a 1.0 M solution of tetrabutylammonium fluoride (0.5 ml, 0.5mmol) was added at room temperature, and the mixture was stirred for 30minutes at the same temperature. The solvent was evaporated underreduced pressure. The residue was applied to a short silica gel column(eluent; ethyl acetate:ethanol=30:1), to thereby yield 0.214 g of acrude compound with no triethylsilyl group.

The above-described compound with no triethylsilyl group intetrahydrofuran (2 ml) and anhydrous ethanol (0.1 ml) were fed to aflask. Ammonia gas was condensed at −78° C. to 20 ml and fed to theflask. Metallic sodium (0.062 g, 2.7 mmol) was added quickly in an argonatmosphere, followed by stirring for 30 minutes at the same temperature.After ammonium chloride was added thereto, the mixture was stirred fortwo hours at room temperature, and ethanol was added to the mixture.Insoluble materials were separated through filtration by use of Celiteand washed with ethanol two times. The resultant filtrate and thewashing liquid were concentrated under reduced pressure. The residue wasapplied to a silica gel column (13 g, eluent; ethyl acetate :methanol=10:1), to thereby yield Compound 28 in an amount of 0.099 g(76%).

¹H-NMR(DMSO-d₆) δ 7.89 (1H, s, H-8), 6.71 (2H, br s, NH₂), 6.20 (1H, t,J=6.3 Hz, H-1′), 5.74 (2H, br s, NH₂), 5.59 (1H, t, J=5.9 Hz, OH), 5.47(1H, d, J=4.9 Hz, OH), 4.50 (1H, q, J=5.9 Hz, H-3′), 3.65 (1H, dd,J=11.7, 5.4 Hz, H-5′), 3.56 (1H, dd, J=11.7, 7.3 Hz, H-5′), 3.46 (1H, s,ethynyl-H), 2.64 (1H, dt, J=12.7, 6.4 Hz, H-2′), 2.32 (1H, dt, J=13.2,6.4 Hz, H-2′).

Synthesis Example 7

Synthesis of 2′-deoxy-4′-C-ethynylinosine (Compound 29)

To a Tris-HCl buffer solution (6 ml, pH 7.5) of Compound 23 (0.022 g,0.08 mmol), adenosine deaminase (0.044 ml, 20 unit) was added, and themixture was stirred for 2.5 hours at 40° C., followed by cooling to roomtemperature. The reaction mixture was applied to a reverse-phase ODSsilica gel column (50 g), desalted by water (500 ml) flow, and throughuse of a 2.5% aqueous ethanol, Compound 29 was eluted. Subsequently, theCompound 29 was pulverized with isopropanol, to thereby yield 0.016 g ofCompound 29 (72%).

¹H-NMR (DMSO-d₆) δ 12.28 (1H, brs, NH), 8.29 (1H, s, purine-H), 8.06(1H, s, purine-H), 6.32 (1H, dd, J=6.8, 4.9 Hz, H-1′), 5.57 (1H, d,J=5.4 Hz, OH), 5.32 (1H, t, J=5.9 Hz, OH), 4.56 (1H, dt, J=6.4, 5.4 Hz,H-3′), 3.65 (1H, dd, J=12.2, 5.9 Hz, H-5′), 3.57 (1H, dd, J=11.7, 6.4Hz, H-5′), 3.50 (1H, s, ethynyl-H), 2.66 (1H, dt, J=12.2, 5.9 Hz, H-2′),2.46 (1H, dt, J=13.2, 6.9 Hz, H-2′).

Synthesis Example 8

Synthesis of 2′-deoxy-4′-C-ethynylguanosine (Compound 30)

To a Tris-HCl buffer solution (7.8 ml, pH 7.5) of Compound 28 (0.03 g,0.103 mmol), adenosine deaminase (0.057 ml, 20 unit) was added, and themixture was stirred for 2 hours at 40° C., followed by cooling to roomtemperature. The reaction mixture was applied to a reverse-phase ODSsilica gel column (50 g), desalted by water (500 ml) flow, and throughuse of aqueous 2.5% ethanol, Compound 30 was eluted. Recrystallizationfrom water yielded Compound 30 in an amount of 0.015 g (50%).

¹H-NMR(DMSO-d₆) δ 10.61 (1H, br s, NH), 7.90 (1H, s, H-8), 6.48 (2H, brs, NH₂), 6.13 (1H, dd, J=7.3, 5.9 Hz, H-1′), 5.51 (1H, d, J=4.9 Hz, OH),5.30 (1H, t, J=5.9 Hz, OH), 4.47 (1H, dt, J=6.4, 5.4 Hz, H-3′), 3.62(1H, dd, J=12.2, 6.4 Hz, H-5′), 3.54(1H, dd, J=12.2, 6.4 Hz, H-5′), 3.47(1H, s, ethynyl-H), 2.56 (1H, dt, J=12.2, 6.4 Hz, H-2′), 2.36 (1H, dt,J=12.7, 6.8 Hz, H-2′).

Synthesis Example 9

Adenine, guanine, and 2,6-diaminopurine were employed instead of uracilused in Synthesis Example 3 (2), and reaction is carried out in a mannersimilar to that described above (amination by use of triazole describedin (5) omitted), to thereby synthesize the following compounds:

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)adenine;

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)guanine; and

9-(4-C-ethynyl-β-D-arabino-pentofuranosyl)-2,6-diaminopurine.

Synthesis Example 10

(1) Synthesis of2′-O-acetyl-3′,5′-di-O-benzyl-4′-C-triethylsilylethynyl-5-fluorouridine(Compound 31)

Compound 6 (2.00 g, 3.62 mmol) was dissolved in 1,2-dichloroethane (60.0ml), and 5-fluorouracil (0.71 g, 5.46 mmol) andN,O-bis(trimethylsilyl)acetamide (5.37 ml, 21.7 mmol) were added to thesolution, followed by refluxing for one hour. After the reaction mixturewas allowed to cool to room temperature, trimethylsilyltrifluoromethanesulfonate (0.85 ml, 4.70 mmol) was added thereto,followed by stirring overnight at 50° C. A saturated aqueous solution ofsodium hydrogencarbonate was added to the mixture, and after stirring,the organic layer was dried over anhydrous magnesium sulfate. Theresidue was purified by means of silica gel column chromatography(silica gel 300 ml, eluent; n-hexane:ethyl acetate=3:1), to therebyyield a colorless viscous compound (Compound 31; 0.80 g, 1.28 mmol,35.4%).

¹H-NMR(CDCl₃) δ 7.86(1H, d, H-6, J_(6, F)=6.35),7.37-7.29(10H, m,aromatic),6.32(1H, dd, H-1′,J=5.62,1.47), 5.17(1H, t, H-2′,J_(2′,3′)=5.62), 4.73, 4.55(each 1H, d, benzyl, J_(gem)=11.72), 4;55,4.50(each 1H, d, benzyl, J_(gem)=11.72), 4.32(1H, d, H-3′,J_(2′,3′)=5.86), 3.87, 3.63(each 1H, d, H-5 D_(gem)=10.50), 2.04(3H, s,acetyl), 0.96(9H, t, Si—CH₂—CH₃, J=8.06), 0.59(6H, Si—CH₂—CH₃, J=7.81).FABMS m/z:623(MH⁺). HRMS m/z (MH⁺): Calcd. for C₃₃H₄₀FN₂O₇Si: 623.2589,Found:623.2589. [α]_(D) −23.3° (c=0.18, CHCl₃).

(2) Synthesis of3′,5′-di-O-benzyl-4′-C-triethylsilylethynyl-5-fluoroouridine (Compound32):

Compound 31 (0.77 g, 1.24 mmol) was dissolved in methanol (45.0 ml), andtriethylamine (5.00 ml) was added to the solution, followed by stirringfor 48 hours at 30° C. The reaction mixture was concentrated throughdistillation under reduced pressure, and the residue was purified bymeans of silica gel column chromatography (silica gel 100 ml, eluent;n-hexane:ethyl acetate=2:1), to thereby yield a white powdery compound(Compound 32; 0.68 g, 1.17 mmol, 94.4%).

¹H-NMR(CDCl₃) δ 8.42(1H, br. s, 3-NH), 7.80(1H, d, J_(6, F)=6.10),7.38-7.29(10H, m, aromatic), 6.10(1H, dd, H-1′, J=5.98,1.47), 5.00,4.63(each 1H, d, benzyl, J_(gem)=11.23), 4.58, 4.54(each 1H, d, benzyl,J_(gem)=10.99), 4.20(1H, m, H-2), 4.13(1H, d, H-3′, J_(2′,3′)=5.86),3.88, 3.70(each 1H, d ,H-5′, J_(gem)=10.25), 2.99(1H, d, 2′-OH, J=9.77),0.96(9H, t, Si—CH₂—CH₃, J=8.06), 0.58(6H, Si—CH₂—CH₃, J=7.82). FABMSm/z:581(MH⁺). HRMS m/z (MH⁺): Calcd. for C₃₁H₃₈FN₂O₆Si: 581.2483,Found:581.2484. [α]_(D) −16.3° (c=1.05, CHCl₃) m.p. 138-139° C.

(3) Synthesis of 4′-C-triethylsilylethynyl-5-fluorouridine (Compound33):

Compound 32 (1.00 g, 1.72 mmol) was dissolved in dichloromethane (50.0ml), and a solution (26.7 ml, 26.7 mmol) of 1.0 M boron trichloride indichloromethane was added thereto at −78° C. in an argon atmosphere,followed by stirring for three hours at the same temperature. A mixtureof pyridine (10.0 ml) and methanol (20.0 ml) was added at −78° C.,followed by stirring for 30 minutes. The reaction mixture wasconcentrated through distillation under reduced pressure, and theresidue was partitioned with ethyl acetate and water. The organic layerwas dried over anhydrous magnesium sulfate and concentrated throughdistillation under reduced pressure. The residue was purified by meansof silica gel column chromatography (silica gel 150 ml, eluent;chloroform methanol=10:1), to thereby yield a white powdery compound

(Compound 33; 0.64 g, 1.60 mmol, 93.0%).

¹H-NMR(DMSO-d₆) δ 11.93(1H, d, 3-NH, J=5.13), 8.13(1H, d, H-6,J_(6, F)=7.08), 5.89(1H, dd, H-1′,J=6.35,1.95), 5.71(1H, t, 5′-OH,J=5.37), 5.37, 5.23(each 1H, d, 2′-OH, 3′-OH, J=6.35), 4.12(1H, q, H-2,J=6.35), 4.05(1H, t, H-3, J=5.61), 3.61-3.57(2H, m, H-5), 3.35(1H, s,ethynyl),0.95(9H, t, Si—CH₂—CH₃, J=7.81), 0.55(6H, Si—CH₂—CH₃, J=7.81).FABMS m/z:401(MH⁺). HRMS m/z (MH⁺): Calcd. for C₁₇H₂₆FN₂O₆Si: 401.1544,Found: 401.1550. [α]_(D) −2.30° (c=1.00, CH₃OH); m.p. 180-183° C.

(4) Synthesis of3′,5′-di-O-acetyl-2′-deoxy-4′-C-triethylsilylethynyl-5-fluorouridine(Compound 34):

Compound 33 (0.54 g, 1.35 mmol) was suspended in acetonitrile (30.0 ml),and a solution (20.0 ml) of acetyl bromide (1.00 ml, 13.5 mmol) inacetonitrile was added dropwise to the suspension at 85° C. over onehour, followed by refluxing for a further three hours. After thereaction mixture was concentrated through distillation under reducedpressure, the residue was dissolved in ethyl acetate and the solutionwas washed sequentially with a saturated aqueous sodiumhydrogencarbonate solution and a saturated aqueous sodium chloridesolution. The organic layer was dried over anhydrous magnesium sulfateand concentrated through distillation under reduced pressure, to therebyyield3′,5′-di-O-acetyl-2′-bromo-2′-deoxy-4′-C-triethylsilylethynyl-5-fluorouridinein crude form (Compound 34). After the crude product (Compound 34) wasconcentrated by co-boiling with toluene three times, the product wasdissolved in dry toluene (20.0 ml). Hydrogenated tri(n-butyl)tin (0.75ml, 2.91 mmol) and 2,2′-azobis(isobutyronitrile) (0.01 g) were added tothe solution at 85° C., and the mixture was heated under stirring for 30minutes in an argon atmosphere. After the reaction mixture wasconcentrated through distillation under reduced pressure, the residuewas purified by means of silica gel column chromatography (silica gel200 ml, eluent; n-hexane:ethyl acetate), to thereby yield a whitepowdery compound (Compound 35; 0.41 g, 0.88 mmol, 65.2%).

¹H-NMR(CDCl₃) δ 9.23(1H, br.s, 3-NH), 7.70(1H, d, H-6, J_(6,F)=6.10),6.35(1H, t, H-1,J_(1′,2′)=7.08), 5.36(1H, t, H-3′, J_(2′,3′)=7.57),4.43, 4.39(each 1H, d, H-5′, J_(gem)=12.21), 2.65, 2.33(each 1H, m,H-2′), 2.17, 2.13(each 3H, s, acetyl), 1.00(9H, t, Si—CH₂—CH₃, J=7.82),0.63(6H, Si—CH₂—CH₃, J=7.82). FABMS m/z:469(MH⁺). HRMS m/z (MH⁺): Calcd.for C₂₁H₃₀FN₂O₇Si: 469.1806, Found: 469.1810. [α]_(D) −12.9° (c=1.00,CHCl₃); m.p. 111-112° C.

(5) Synthesis of 4′-C-ethynyl-2′-deoxy-5-fluorocytidine

(Compound 37)

Compound 35 (0.35 g, 0.75 mmol) was dissolved in pyridine (5.00 ml), andp-chlorophenylphosphrodichloridate (0.62 ml, 3.77 mmol) was added to theresultant solution under ice-cooling, followed by stirring for fiveminutes. 1,2,4-Triazole (0.78 g, 11.3 mmol) was added to the mixture,followed by stirring for 24 hours at 30° C. The reaction mixture wasconcentrated through distillation under reduced pressure, and theresidue was partitioned with ethyl acetate and water. The organic layerwas dried over anhydrous magnesium sulfate, and concentrated throughdistillation under reduced pressure. The residue was purified by meansof silica gel column chromatography (silica gel 50 ml, eluent; ethylacetate), to thereby yield a colorless viscous compound(4′-C-triethylsilylethynyl-2′-deoxy-5-fluoro-4-(1,2,4-triazolo)uridine;Compound 36). Compound 36 was dissolved in dioxane (15.0 ml), and 25%aqueous ammonia (5.00 ml) was added to the resultant solution, followedby stirring overnight at room temperature. After disappearance ofCompound 36 had been confirmed by means of silica gel thin-layerchromatography (chloroform:methanol=10:1), the reaction mixture wasconcentrated through distillation under reduced pressure. The residuewas dissolved in methanol (45.0 ml), and an aqueous 1 N sodium hydroxidesolution (5.00 ml, 5.00 mmol) was added thereto, followed by stirringfor 24 hours at room temperature. Acetic acid (0.29 ml, 5.00 mmol) wasadded to the mixture, and the reaction mixture was concentrated throughdistillation under reduced pressure. The residue was purified by meansof silica gel column chromatography (silica gel 50 ml;chloroform:ethanol=4:1). The fraction containing Compound 37 was broughtto dryness under reduced pressure, and the residue was crystallized frommethanol-ether, to thereby yield a white crystalline compound

(Compound 37; 0.12 g, 0.45 mmol, 60.0%).

¹H-NMR(DMSO-d₆) δ 8.06(1H, d, H-6, J_(6,F)=7.08), 7.79, 7.54 (each 1H,br.s, NH₂), 6.05(1H, m, H-1′), 5.57, 5.50(each 1H, br, 3′-OH, 5′-OH),4.31(1H, br.q, H-3), 3.66, 3.60(each 1H, d, H-5, J_(gem)=11.72),3.51(1H, s, ethynyl), 2.25, 2.12(each 1H, m, H-2′); [α]_(D) +77.9°(c=1.00, CH₃OH); FABMS m/z:270(MH⁺). HRMS m/z (MH⁺): Calcd. forC₁₁H₁₃FN₃O₄: 270.0890, Found:270.0888. m.p. ˜225° C. (December).

Drug Preparation Example 1: Tablets Compound of the present invention30.0 mg Cellulose micropowder 25.0 mg Lactose 39.5 mg Starch 40.0 mgTalc 5.0 mg Magnesium stearate 0.5 mg Tablets are prepared from theabove composition through a customary method.

Drug Preparation Example 1: Tablets Compound of the present invention30.0 mg Cellulose micropowder 25.0 mg Lactose 39.5 mg Starch 40.0 mgTalc 5.0 mg Magnesium stearate 0.5 mg Tablets are prepared from theabove composition through a customary method.

Drug Preparation Example 3: Injections Compound of the present invention30.0 mg Glucose 100.0 mg

Injections are prepared by dissolving the above composition in purifiedwater for preparing injections.

Test Examples will next be described. Employed in tests were thefollowing seven compounds of the present invention and two knowncompounds:

Compound 13: 4′-C-ethynyl-2′-deoxycytidine;

Compound 19: 1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)cytosine;

Compound 23: 9-(2-deoxy-4-C-ethynyl-β-D-ribo-pentofuranosyl)adenine(4′-C-ethynyl-2′-deoxyadenosine);

Compound 28: 9-(2-deoxy-4-C-ethynyl-β-D-ribo-pentofuranosyl)-2,6-diaminopurine;

Compound 29: 9-(2-deoxy-4-C-ethynyl-β-D-ribo-pentofuranosyl)hypoxanthine(4′-C-ethynyl-2′-deoxyinosine);

Compound 30: 9-(2-deoxy-4-C-ethynyl-β-D-ribo-pentofuranosyl)guanine(4′-C-ethynyl-2′-deoxyguanosine);

Compound 37: 4′-C-ethynyl-2′-deoxy-5-fluorocytidine); and

Known compounds: 4′-C-ethynylthymidine and AZT.

Test Examples

<Test methods>

(1) Anti-HSV-1 activity

1. Human embryonic lung cells are subcultured by splitting at 1:2 to 1:4in an Eagle's MEM supplemented with 10% bovine serum (MitsubishiChemical Corporation) at intervals of 4-5 days.

2. The suspension of cells obtained from parent cells by splitting at1:2 is added to a 96-well-microplate at 200 μl/well, and the cells arecultured in a CO₂-incubator for four days at 37° C.

3. After culture medium is removed, a test agent (100 μl) in serialfivefold dilution with a Hanks' MEM is added to the wells.

4. An Eagle's MEM (100 μl) supplemented with 5% bovine serum containing100-320 TCID₅₀ of herpes simplex virus type-1, VR-3 strain is added tothe wells to thereby seed the virus, and the infected cells are culturedat 37° C. in a CO₂-incubator.

5. After the cells are cultured for 2-3 days, the degree of CPE of eachwell is observed under a microscope for evaluation on a scale of 0 to 4.When the cells in test agent-free controls are completely degeneratedthrough infection with the virus, the CPE score is 4.

6. The antiviral activity is expressed as ED₅₀ at which HSV-induced CPEwere expressed at least 50%.

(2) Anti-human immunodeficiency virus (HIV) activity

1) MTT method using MT-4 cells

1. A test agent (100 μl) is diluted on a 96-well microplate. MT-4 cellsinfected with HIV-1 (III_(b) strain; 100 TCID₅₀) and non-infected MT-4cells are added to the microplate such that the number of cells in eachwell becomes 10,000. The cells are cultured at 37° C. for five days.

2. MTT (20 μl, 7.5 mg/ml) is added to each well, and the cells arefurther cultured for 2-3 hours.

3. The cultured medium (120 μl) is sampled, and MTT terminating solution(isopropanol containing 4% Triton X-100 and 0.04N HCl) is added to thesample. The mixture is stirred to form formazane, which is dissolved.The absorbance at 540 nm of the solution is measured. Since theabsorbance is proportional to the number of viable cells, the test agentconcentration at which a half value of the absorbance is measured in atest using infected MT-4 cells represents EC₅₀, whereas the test agentconcentration at which a half value of the absorbance is measured in atest using non-infected MT-4 cells represents CC₅₀.

2) MAGI assay using HeLa CD4/LTR-beta-Gal cells

1. HeLa CD4/LTR-beta-Gal cells are added to 96 wells such that thenumber of cells in each well is 10,000. After 12-24 hours, the culturemedium is removed, and a diluted test agent (100 μl) is added.

2. A variety of HIV strains (wild strain: WT, drug-resistant strain:MDR, M184V, NL4-3, 104pre, and C; each equivalent to 50 TCID₅₀) areadded, and the cells are further cultured for 48 hours.

3. The cells are fixed for five minutes using PBS containing 1%formaldehyde and 0.2% glutaraldehyde.

4. After the fixed cells are washed with PBS three times, the cells arestained with 0.4 mg/ml X-Gal for one hour, and the number ofblue-stained cells of each well is counted under a transmissionstereoscopic microscope. The test agent concentration at whichblue-stained cells decrease to 50% and 90% in number represents EC₅₀ andEC₉₀, respectively.

5. In a manner similar to that employed in the MTT method, cytotoxicityis measured by use of HeLa CD4/LTR-beta-Gal cells.

The test results are shown in Tables 1 to 7.

<Results>

(1) Anti-HSV-1 Activity

TABLE 1 Drug HSV-1 (ED₅₀, μg/ml) Compound 13 33

(2) Anti-human Immunodeficiency Virus (HIV) Activity and Cytotoxicity

Each value shown in Tables 2 to 7 represents an average of two to fiveassayed values.

1. MTT method using MT-4 cells

TABLE 2 MT-4 cells HIV-1 Cytotoxicity Drugs (EC₅₀, μg/ml) (CC₅₀, μg/ml)Compound 13 0.0012 0.56 Compound 19 0.0115 0.53 4′-C-ethynyl 0.22 >100thymidine AZT 0.0016 >0.27

TABLE 2 MT-4 cells HIV-1 Cytotoxicity Drugs (EC₅₀, μg/ml) (CC₅₀, μg/ml)Compound 13 0.0012 0.56 Compound 19 0.0115 0.53 4′-C-ethynyl 0.22 >100thymidine AZT 0.0016 >0.27

TABLE 4 MT-4 cells HIV-1 Cytotoxicity Drugs (EC₅₀, μM) (CC₅₀, μM)Compound 37 0.033 >500 AZT 0.055

TABLE 4 MT-4 cells HIV-1 Cytotoxicity Drugs (EC₅₀, μM) (CC₅₀, μM)Compound 37 0.033 >500 AZT 0.055

TABLE 4 MT-4 cells HIV-1 Cytotoxicity Drugs (EC₅₀, μM) (CC₅₀, μM)Compound 37 0.033 >500 AZT 0.055

2. MAGI assay using HeLa CD4/LTR-beta-Gal cells

TABLE 7 HeLa CD4/LTR-beta-Gal cells HIV 104 pre EC₅₀, μM Drugs NL-43(EC₉₀, μM) C Compound 37 0.021 0.022  0.122 (0.25 0.19  3.44) AZT 0.1090.059  3.269 (4.96 9.66 >10)

What is claimed is:
 1. A compound which is selected from the groupconsisting of 4′-C-ethynyl-2′-deoxycytidine,4′-C-ethynyl-2′-deoxy-5-fluorocytidine, and1-(4-C-ethynyl-β-D-arabino-pentofuranosyl)cytosine, or a 5′-phosphateester thereof, or a pharmaceutically acceptable salt, hydrate, orsolvate thereof.
 2. The compound according to claim 1, which is4′-C-ethynyl-2′-deoxycytidine.
 3. The compound according to claim 1,which is 4′-C-ethynyl-2′-deoxy-5-fluorocytidine.
 4. The compoundaccording to claim 1, which is1-(4-C-ethynyI-β-D-arabino-pentofuranosyl)cytosine.
 5. A pharmaceuticalcomposition comprising the compound according to claim 1 and apharmaceutically acceptable carrier.
 6. A pharmaceutical compositioncomprising the compound according to claim 2 and a pharmaceuticallyacceptable carrier.
 7. A pharmaceutical composition comprising thecompound according to claim 3 and a pharmaceutically acceptable carrier.8. A pharmaceutical composition comprising the compound according toclaim 4 and a pharmaceutically acceptable carrier.